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Cell Isolation from Tissue
1.44
Cell Isolation from Tissue
MR Mirbolooki, H Bozorgmanesh, C Foster III, W Kuhtrieber, and JRT Lakey, University of California at Irvine, Orange, CA, USA © 2011 Elsevier B.V. All rights reserved.
1.44.1 1.44.2 1.44.3 1.44.4 1.44.5 1.44.6 1.44.6.1 1.44.6.2 1.44.6.2.1 1.44.6.2.2 1.44.6.2.3 1.44.6.2.4 1.44.6.2.5 1.44.6.2.6 1.44.6.2.7 1.44.6.2.8 1.44.6.2.9 1.44.6.2.10 1.44.7 1.44.7.1 1.44.7.2 1.44.7.3 1.44.7.4 1.44.8 1.44.8.1 1.44.8.2 1.44.8.3 1.44.9 References
Introduction Tissue/Organ Procurement Tissue/Organ Preservation Tissue/Organ Rinsing Tissue/Organ Fragmentation Cell Dissociation Nonenzymatic Dissociation Enzymatic Dissociation Stromal cells Adipocytes Thymus and umbilical cord Breast tissue Colorectal cancer cells Epithelial cells from intestine Hepatocytes Cells from skin Hair follicles Synovia and cartilage Purification Regular Centrifugation Gradient Centrifugation Adherence Fluorescence-Activated Cell Sorting Cell Yield, Viability, and Purity Assessment Cell Yield Cell Viability Purity Conclusions
Glossary cell isolation Separation of certain cells from a tissue/ organ. cytotoxicity The degree to which something is toxic to living cells. endonuclease An enzyme that cleaves the phosphodiester bond within a polynucleotide chain. lyophilized Dried by freezing in a high vacuum.
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organ perfusion Injection of a fluid into a blood vessel of the procured organ or tissues. organ preservation Transporting of donor organs, after surgical removal, for processing and transplant. organ procurement Obtaining organs for transplantation or cell isolation. reconstitution To bring back a liquid in a concentrated or powder form to its normal strength by adding water.
1.44.1 Introduction Over the past decades, interest has grown in identifying and characterizing cells isolated from a range of types of human tissues and organs. Experimental models and clinical trials have provided great opportunities to investigate the remarkable biological and clinical properties of these cells. For instance, stromal cells [1] found in many adult tissues have been an attractive stem cell source for the regeneration of damaged tissues in clinical applications; cultured skin substitutes (CSS) [2] composed of dermal fibroblasts and epidermal keratinocytes have been used as an adjunctive therapy in the treatment of large burn wounds; and transplantation of limbal tissue has replenished the stem cells population to support the regeneration of the entire corneal surface epithelium [3]. Isolation of cells from organs such as liver and pancreas has been in the center of interest in many research laboratories. The
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engraftment of transplanted hepatocytes in the liver has drawn significant interest from the field of cell therapy [4], and islet transplantation has shown the potential to prevent chronic pancreas transplantation complications in diabetics, while providing physiologic glucose control [5]. In spite of the advances in the field, there is a confusing inconsistency in the methods of cell isolation and they seem to be far from optimum. Therefore, the choice of one technique over another has often been arbitrary and based more on individual experiences rather than on an understanding of why a certain method works and what modifications could lead it to an even better outcome. The main goal of a cell isolation procedure is to optimize the yield of functionally viable, isolated cells. Many factors affect cell isolation, as it is a complex procedure. Type of tissue, donor’s factors such as body mass index (BMI) and age, warm/cold ischemia during organ procurement/preservation, isolation solution, enzyme(s), variances in any enzyme preparation, concentra tion(s) of enzyme(s), and digestion time are among the factors that affect the cell isolation outcome. Scientists searching the literature for the protocols for using digestive enzymes and optimal conditions for tissue/cell isolation are often confronted with conflicting data. As a first step, it is important to develop a consistent and reproducible method of isolating intact and viable cells. This article summarizes different tissue/cell isolation protocols to achieve a logical approach for establishing a specific cell isolation standard operating procedure.
1.44.2 Tissue/Organ Procurement Many factors limit the success of a cell isolation procedure. Donor factors such as cause of death [6] and donor age [7], along with the surgical procurement procedure, are among the key factors to be considered at the time of tissue/organ acceptance for the purpose of cell isolation and transplantation. If perfusion is not required to obtain the organ/tissue, the procedure is relatively simple and easy. Tissues are procured either from cadavers or from live donors who are undergoing surgery for another reason. Human subcutaneous adipose tissue can be obtained from healthy women undergoing abdominal dermolipectomy for plastic surgery [6]. Bone marrow aspirates can be obtained from the femoral shaft of patients undergoing total hip replacement or puncturing the iliac crest at an orthopedic department [7]. Even discarded tissue like human thymus can be obtained from young children undergoing corrective cardiac surgery [8]. However, if perfusion is required to obtain an organ like liver or pancreas, the procedure is more complicated and many factors including warm/cold ischemia could affect the outcome of cell isolation. The human pancreas is one of the most challenging organs to procure for vascularized clinical transplantation, based on a need to preserve the integrity of its capsule and of the vascular pedicles for implantation while avoiding injury to the inflow to the liver [9]. Different challenges persist when the pancreas is procured for the purpose of islet isolation, because precise procurement techniques are essential for successful isolation of large numbers of viable islets. Surgical expertise, procurement technique, and minimal ischemia time have a major impact on the recovery of functionally viable islets and posttransplant clinical outcomes. In these cases, the donor organs are typically perfused in situ with cold University of Wisconsin (UW) solution before explantation. Cold ischemia time during procurement is defined as the time after clamping of aorta until excision of the organ, which varies from 20 min to 4 h. In some cases, the organ is placed on ice and perfused with ice-cold UW solution by means of multiple catheters inserted into the vessels on the cut surface of the resected fragment immediately after excision [10]. Although the most common perfusion solution is UW in multiple organ procurement, perfusion can be done with other solutions including the simple option of Hanks’ balanced salt solution (HBSS) lacking calcium and magnesium and containing 10 mM HEPES and 5 mM ethyleneglycoltetraacetic acid (EGTA) [11].
1.44.3 Tissue/Organ Preservation During organ procurement, blood supply and hence oxygen supply is necessarily interrupted. Therefore, cells cannot continue to meet the energy demands of the active ion-transporting systems and this leads to cell death [12]. Research efforts by one of the pioneers of organ preservation Folkert O. Belzer and his colleague James H. Southard resulted in the development of a preservation solution in the late 1980s based on five philosophies [13]. They developed a UW solution containing impermeants (raffinose, lactobionate) to minimize hypothermia-induced cell swelling, buffers (phosphate) to prevent intracellular acidosis, a colloid (hydroxyethyl starch) to prevent the expansion of interstitial space during the flush-out period, free radical inhibitors and scavengers (glutathione, allopurinol) to prevent injury from oxygen free radicals during ischemia and after reperfusion, and energy precursors (Mg+, adenosine) for energy metabolism during reperfusion period. Histidine–tryptophane–ketoglutarate (HTK) solution is another common preservation solution. It was developed in the 1970s by Bretschneider as a cardioplegia solution [14] and is being used increasingly for both kidney [15] and liver [16] transplantation. HTK contains less potassium and sodium and a strong histidine buffer that increases the osmotic effect of mannitol, which is also included in this solution. Tryptophan is added as a membrane stabilizer, and ketoglutarate is added as a metabolism substrate. These solutions are designed for long-term (12–24 h) organ preservation. For shorter preservation, the media can be simple. Endometrial tissue, for instance, is transported to the laboratory in an isolation medium consisting of Dulbecco’s modified Eagle medium, high glucose (DMEM-H) culture medium and 5% fetal bovine serum (FBS) plus antibiotics [17].
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1.44.4 Tissue/Organ Rinsing The tissue pieces may need to be washed by an appropriate buffer like phosphate buffered saline (PBS) [7] or Hank’s buffered saline solution (HBSS) [18, 19] to remove contaminating debris and red blood cells. The washing solution may also contain antibiotics [18, 19]. For some tissues, 2.5 µg/ml amphotericin B [20] is added to cover a broad range of bacteria. The washing step usually takes 5–10 min; however, tissue may be incubated in buffer for a longer time (~30 min) to more efficiently reduce contamination of the tissue with blood cells and soluble factors [21]. Tissue may even be disinfected and dissociated at the same time in DMEM containing penicillin, streptomycin, and collagenase [19]. In some specific tissues like colonic mucosal speci mens, samples are thoroughly washed in PBS containing 100 IU ml−1 penicillin–streptomycin and 2 mM dithiothreitol (DTT, wash buffer) to remove debris [22]. They might also be washed in 6.5 mM DTT to remove mucus contamination. After gentle removal of the DTT solution, tissue fragments are rinsed once with HBSS [23]. The washing solution may be supplemented by 10% FBS and higher concentrations of antibiotics (200 IU ml−1) for prostate tissues [24]. For certain tissues including the brain ventricular zone [25], intestinal mucosa [26], and umbilical cord [27] tissues, washing solution is Ca/Mg-free HBSS. The corneoscleral tissue (HCECs) is rinsed three times with DMEM containing specific antibiotics (50 mg/ml gentamicin and 1.25 mg ml−1 amphotericin B) [28].
1.44.5 Tissue/Organ Fragmentation Generally, the tissues need to be cut into multiple pieces with sterile scissors or scalpel [18]. The size of the pieces varies based on the tissue type and the purpose of cell isolation. Whereas adipose tissue was cut into small pieces with average weight of 20–50 mg in one study [6], the same tissue was cut into 10–20 mg pieces in another study [21]. In some cell isolation procedures like breast cancer cell isolation, the tissue is cut into small pieces, and the pieces are then minced with a blade to yield 2–3 mm3 pieces [29]. In some organs like pancreas, the tissue is fragmented into larger pieces after enzyme perfusion.
1.44.6 Cell Dissociation 1.44.6.1
Nonenzymatic Dissociation
Cells attach to surfaces and to each other by cell surface adhesion molecules. Cell adhesion molecules (CAMs) can be classified into four major families [30]: cadherins, integrins, selectins, and immunoglobulin (Ig) CAMs. The first three are calcium and/or magnesium dependent, whereas members of the last group do not require divalent ions. Removal of calcium and magnesium causes the cells to dissociate from the surface and/or from each other. Extracellular calcium is also needed for the formation of tight junctions between cells [31]. Nonenzymatic cell dissociation media do not contain divalent ions and are often supplemented with mixtures of chelators that remove additional divalent ions from the tissues. The commonly used chelators are ethylenediaminetetraacetic acid (EDTA), EGTA, and citrate. These chelators can bind calcium and magnesium from various CAMs. Chelating agents are dissolved at concentrations of 0.1–2% in Ca/Mg-free HBSS or PBS. The solutions may also contain stabilizing agents such as glycerol. Several companies market proprietary nonenzymatic dissociation solutions and claim that they are more effective as compared to the simple chelator-containing solutions. Well-known products are from Sigma, ATCC, Cellgro (Mediatech), Millipore, and Invitrogen. Nonenzymatic dissociation solutions can typically be stored for long periods of time at room temperature. Cytotoxicity of nonenzymatic solutions is highly dependent on the cell types being treated [32]. While on the one hand it is known that chelators such as EDTA can have cytotoxic effects, on the other hand, cells can be exposed for longer periods of time to such solutions as compared to trypsin without the risk of damage associated with protein overdigestion.
1.44.6.2
Enzymatic Dissociation
A major obstacle to successful cell dissociation has been the inconsistent enzymatic activity and stability of the enzyme prepara tions. Variability in enzyme blends has been considered the most important determinant of the success or failure in isolated cell yields, and this variation in potency has been observed between, and even within, enzyme lots [33]. Enzymes are typically available as lyophilized powders. They may be stored at 2–8 °C, and special care is required when opening the enzyme vials. They should not be opened in humid areas. Any vial has to be brought to room temperature before opening. Ideally, the vials should be taken from the refrigerator at least a half hour before opening, and should be left in a dessicator. Before opening the vials, it must be made sure that it is not at all cool to the touch. Once diluted with media or buffer, proteolytic enzymes can undergo autolysis; hence enzymes should be dissolved only just prior to use. Reconstituted enzymes should not be stored at 2–8 °C; if necessary, they can be aliquoted and frozen at –20 °C. Repeated freeze–thaw cycles should be avoided. All enzymes, upon reconstitution, can be sterile filtered through a 0.22-µm-pore-size membrane. As there are variations in the isolation procedures of different types of cells, we will address the differences in enzymes usage, digestion time, enzyme blockade, and collection media between various types of cells isolated from the tissues.
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1.44.6.2.1
Stromal cells
Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside. Stromal cells can be isolated from a variety of tissues, such as bone marrow, periosteum, trabecular bone, synovium, skeletal muscle, deciduous teeth, and adipose tissues. Collagenase type I (from Clostridium histolyticum) is a crude collagenase preparation that can be used for the isolation of stromal cells. The preparation contains average amounts of caseinase, clostripain, and tryptic activities. To separate the human adipose-derived stromal cell (ASC) fraction from adipocytes, Traktuev et al. digested the tissues in collagenase type I solution under agitation for 2 h at 37 °C and centrifuged at 300 g for 8 min. They resuspended the pellet in DMEM–Ham’s Nutrient Mixture F12 (DMEM–F12) containing 10% FBS, filtered it through 250-μm filters and centrifuged at 300 g for 8 min [1]. There are different versions of stromal cells isolation protocols available in the literature with minor differences. For instance, adipose tissues are treated with collagenase type I (1 mg ml−1 in HBSS with 1% bovine serum albumin (BSA)) for just 60 min at 37 ° C with intermittent shaking [34] or for 30–60 min at the same temperature with gentle agitation [7]. The pellet is centrifuged at the same g-force (300 g) but for a shorter time (5 min) [34] or at a higher g-force (400 g) and for a longer time (10 min) [20]. The activity of the collagenase is neutralized with DMEM-LG containing 10% fetal calf serum (FCS) [7]. The pellets are resuspended in a red blood cell lysis buffer (2.06 g l−1 Tris base, 7.49 g l−1 NH4Cl, pH 7.2) for 10 min at room temperature. The suspended cells are passed first through 100-µm and then through 40-µm cell sieves [20]. The prostatic stromal cells are also digested with collagenase type I (2 mg ml−1) for 2.5 h at 37 °C on a shaking rotor. The tissue digest is vigorously pipetted and epithelial clumps settled from stromal cells for 15 min without centrifugation [24].
1.44.6.2.2
Adipocytes
Adipocytes are traditionally isolated with collagenase type II at different concentrations based on the tissue’s site. It is another form of the purified collagenase enzymes prepared to contain higher clostripain activity. Collagenase type I is also used in adipocyte dissociation. The orbital preadipocyte is digested with collagenase type I (2 mg ml−1) in HBSS for 45 min at 37 °C. The samples are centrifuged at 500 g for 1 min, and the supernatant containing connective tissue debris, collagenase, and lipid is removed leaving a pellet containing preadipocytes [18]. However, it is digested in 1 mg ml−1 collagenase type II, filtered (with 150-µm nylon mesh), and centrifuged at 200 g for 35 s. In some experiments, adipocytes are digested at 37 °C in PBS containing 2% BSA and 2 mg ml−1 collagenase for 45 min and filtered through 25 µm filters [35]. In others, adipose tissue explants are digested in DMEM containing 0.5 mg ml−1 collagenase type II and 1% BSA for 30–40 min at 37 °C under constant shaking. At the end of the incubation period, the reaction is stopped by dilution with DMEM and filtered on a silk screen in order to retain undigested explants [6]. Adipose tissue may even be dissociated for just 5–10 min in DMEM containing antibiotics, 2 mg ml−1 collagenase, and 20 mg ml−1 BSA [19].
1.44.6.2.3
Thymus and umbilical cord
Collagenase type II is also employed for thymus and umbilical cord dissociations. Thymus is cut into small fragments, suspended in 10 ml RPMI 1640 (Rosewell Park Memorial Institute) medium containing 2% FCS, collagenase (1 mg ml−1, type II), and deoxyr ibonuclease (DNAse) (0.02 mg ml−1, grade II bovine pancreatic DNAse I), and then digested with intermittent agitation for 15 min at 37 °C followed by 5 min at room temperature with constant agitation. To disrupt the dendritic cell (DC)–T cell complexes, EDTA is added (to 0.01 M final) to the digest, and incubation with agitation is continued for another 5 min. The suspension is then passed through a stainless-steel sieve to remove aggregates and stromal material [8]. Human umbilical cord segments are washed and flushed with Ca/Mg-free PBS to remove clotted blood. Sixty milliliters of a 0.1% solution of collagenase type II dissolved in Ca/Mg free PBS are gently infused into the umbilical vein and incubated at 37 °C for 20 min. The collagenase–endothelial cell suspension is then allowed to drain slowly into a 50-ml tube. This tube is centrifuged for 10 min at 1000 250 g [27].
1.44.6.2.4
Breast tissue
Breast tissue is digested by collagenase type III with very different concentrations. It is lower in secondary proteolytic contaminant activities but contains typical collagenase activity. After washing with HBSS twice, minced tissue is dissociated with 200 IU ml−1 of collagenase type III in Medium 199 at 37 °C for about 2 h. During incubation, tissue is pipetted every 30 min. Dissociation is stopped by adding 5% FBS, and the cells are diluted with Medium 199 and then filtered sequentially through a sterile 100-μm nylon mesh and a 40-μm cell strainer to obtain a single cell suspension. Cells are then washed twice with HBSS and 2% heat-inactivated calf serum [29]. Breast biopsies are cut and rotated for 24 h in a serum-free medium, DMEM–F12 supplemented with 2 mM glutamine and 50 µg ml−1 gentamicin, and a high concentration of collagenase type III (900 IU ml−1). The fibroblasts are isolated by differential centrifugation of the collagenase digest and plated in DMEM–F12 in T-25 flasks 37 °C.
1.44.6.2.5
Colorectal cancer cells
To separate colorectal cancer cells, collagenease type III is also used in combination with DNase I. DNase I is an endonuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide. After gentle removal of the DTT solution, tissue fragments are rinsed once with HBSS, resuspended in serum-free RPMI medium 1640 (2 mM L-glutamine, 120 µg ml−1 penicillin, 100 µg ml−1 streptomycin, 50 µg ml−1 ceftazidime, 0.25 µg ml−1 amphotericin-B, 20 mM HEPES) with 200 IU ml−1 collage nase type III and 100 IU ml−1 DNase I, and incubated for 2 h at 37 °C to obtain enzymatic disaggregation. Cells are then resuspended by pipetting and serially filtered by using sterile gauze, and 70- and 40-µm nylon meshes [23].
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Epithelial cells from intestine
Epithelial cells are dissociated by collagenase type IV, which has low tryptic activity. This type of collagenase is usually used when the receptors need to remain integrated. The mucosal layer of intestinal specimens is cut into small pieces and treated with 0.1 mM (small intestine) or 0.2 mM (large intestine) DTT with shaking to free the cells from the tissue. The supernatant containing epithelial cells from the luminal/villous compartment and intraepithelial lymphocytes is collected. The remaining tissue pieces are treated subsequently with 72.5 IU ml−1 of collagenase type IV in heat-inactivated human AB serum >with vigorous shaking at 37 °C for 30 min. The cell suspension is then passed through a stainless-steel sieve, resulting in a cell fraction containing epithelial cells mainly from the crypt compartment, lamina propria leucocytes, and stromal cells. Cell fractions are then washed in Tris-buffered HBSS containing 0.2% human serum albumin [36].
1.44.6.2.7
Hepatocytes
Both collagenase types I and IV have been used to isolate hepatocytes. Following 30 min of initial perfusions, the warmed liver is perfused at a flow rate of 60 ml min−1 [11] or 100 ml min−1 [10] with 1.7 l of HBSS (containing calcium and magnesium) supple mented with 0.5% BSA, 0.05% collagenase (type IV), penicillin, and streptomycin. Following the collagenase perfusion, softened sections of the liver are dissected and placed into a sterile beaker and chopped with scissors, and then 500 ml of HBSS containing 0.5% BSA, 0.02% collagenase type IV [11] or 0.05% collagenase type I [10], penicillin, and streptomycin is added to the mixture. The tissue is incubated at 37 °C for 10 min with gentle shaking, and the released cells are filtered first through sterile gauze and then through a 250-µm nylon mesh and are collected into 250-ml centrifuge bottles [11]. Hepatocytes are pelleted by centrifugation at either 500 g for 5 min at 4 °C [37] or 50 g for 3 min and are washed twice with HBSS containing 0.5% BSA, penicillin, and streptomycin [11].
1.44.6.2.8
Cells from skin
Dispase, a neutral protease, has been proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis. First, the dermis and epidermis of the skin are enzymatically separated by incubation with dispase for 2.75 h. The dermal strips are placed in endothelial cell growth medium supplemented with 10% FBS and scraped with sterile angled scissors to release microvascular endothelial cells (HDMEC). The cell suspension is centrifuged and the HDMEC are inoculated in endothelial cell growth medium into flasks coated with attachment factor. At this time collagenase may be added for inducing further cell dissociation. The scraped dermal tissue strips are minced and incubated for 1 h with collagenase for fibroblast isolation [2]. The epidermal pieces are digested in trypsin–EDTA solution and the keratinocytes are inoculated into flasks containing lethally irradiated NIH 3T3 cells in a keratinocyte growth medium. Trypsin enzyme degrades protein and it is often referred to as a proteolytic enzyme. The primary basal keratinocytes can be isolated from neonatal foreskins in 4 mg ml−1 of dispase and 3 mg ml−1 of collagenase in PBS at 37 °C for 1.5 h, and the resultant cell suspension is filtered through a 70-μm cell strainer [38].
1.44.6.2.9
Hair follicles
Hair follicles are also isolated by dispase from scalp tissues (0.5–2 cm2 or less). Tissues are rinsed, trimmed to remove excess adipose tissues, cut into small pieces, and subjected to enzymatic dissociation in 12.5 mg ml−1 dispase in DMEM for 24 h at 4 °C. After treatment, the epidermis is peeled off from the dermis, and hair follicles are plucked from the dermis. Hair follicles are rinsed thoroughly with PBS to prevent contaminating epidermal or dermal cells and are examined under an inverted microscope [39].
1.44.6.2.10
Synovia and cartilage
Synovia are minced and digested with 1.5 mg ml−1 collagenase–dispase, 1 mg ml−1 hyaluronidase, and 0.15 mg ml−1 DNase I for 3–4 h at 37 °C [40]. The septal cartilage specimens are minced into 1- to 3-mm3 cubes, placed into a spinner flask, and incubated at 37 °C in a digestion medium (collagenase type II (2.00 mg ml−1), hyaluronidase (0.10 mg ml−1), and DNase I (0.15 mg ml−1) in DMEM–F12 (1:1) medium) for 18–36 h. After digestion, the dispersed cells are filtered through a 40-μm nylon cell strainer to remove any remaining undigested clumps. The cells are then suspended with PBS and centrifuged at a low speed (1000 g for 7 min) twice to remove any remaining enzymes [41].
1.44.7 Purification It is believed that there are several advantages in transplanting highly purified isolated cells, including improved engraftment, increased safety, and reduced graft immunogenicity [42]. Even in research settings, purified cells provide more consistent and reliable data. The purification of cells from tissue extract is performed in four different ways.
1.44.7.1
Regular Centrifugation
Some cells are purified by just simple centrifugation. Preadipocytes [43] are centrifuged at 90 g for 1 min, and the top adipocyte layer is removed and then centrifuged again at 90 g for 5 min. The pellet containing preadipocytes is removed, and the cells are washed with DMEM–F12. Isolated stromovascular (SV) cells are separated from adipocytes and the medium by centrifugation in 15-ml tubes very gentle for 1 min at 400 g [21] or stronger for 10 min at 600 g [35]. The SV cells are defined as those cells isolated by
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collagenase digestion that do not float. After liver digestion, cell suspension is centrifuged three times in low gravitational force (50 g, 5 min) to separate hepatocytes from nonparenchymal cells [10].
1.44.7.2
Gradient Centrifugation
SV Cell suspensions (15 ml) are applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes. After centrifugation (400 g, 30 min [20] or 8–10 min [17]), the cells at the gradient interface are collected, washed in HBSS, and passed through a 30-µm mesh [20]. The bone marrow aspirates are diluted 1:5 with 2 mM EDTA–PBS. The mononuclear cell (MNC) fraction is isolated by density gradient centrifugation at 435 g for 30 min at room temperature using Ficoll-Hypaque Plus solution [44, 45]. Currently, the purification of islets from exocrine tissue is performed by continuous Ficoll gradients using a refrigerated COBE 2991 cell processor [46]. The cells are recovered from the digest by centrifugation. Then the pellet is immediately resuspended in Nycodenz medium (1.068 g cm−3 and iso-osmotic with human serum), and a low-density fraction is collected after centrifugation at 1700 g for 10 min [8].
1.44.7.3
Adherence
The cellular pellet is washed with DMEM–F12 containing 15% (v/v) FCS and seeded on 48-well plates. Cells are left overnight to attach and all unattached cells (including red blood cells) are washed the following day with HBSS [44, 45]. Nonadherent cells are removed 12–18 h after initial plating by intensely washing the plates. Adipose tissue (AT)-derived fibroblastoid adherent cells are harvested at subconfluence using trypsin [7].
1.44.7.4
Fluorescence-Activated Cell Sorting
The fluorescene-activated cell sorter is a machine that can rapidly separate the cells in a suspension on the basis of size and the color of their fluorescence. This apparatus can sort as many as 300 000 cells per minute. For dendritic cell purification, the cells are then incubated for 25 min with a mixture of monoclonal antibodies (mAbs), including anti-CD3, anti-CD8, anti-CD7, anti CD15, anti-CD19, anti-CD20, and anti-glycophorin A, in EDTA–SS containing 2% human serum. After incubation, the cells coated with mAbs are removed by two cycles of sheep anti-mouse immunoglobulin-coupled magnetic beads. The first cycle is at a 3:1 and the second at a 6:1 bead-to-cell ratio. The cells are then kept overnight at 4 °C in EDTA–SS containing 10% FCS. The next morning, the cells are incubated for 25 min at 4 °C with Cy5-conjugated anti-HLA-DR and biotinylated anti-CD11b in EDTA–SS containing 2% human serum. After two washes, the cells are incubated with streptavidin–Texas Red. DC populations are then sorted by means of a FacStar Plus [8].
1.44.8 Cell Yield, Viability, and Purity Assessment 1.44.8.1
Cell Yield
Cell yield can be estimated by counting trypan blue-stained samples, using a hemocytometer [11, 37]. However, total colonocyte yield is determined as the final wet weight of the purified cell pellet as compared to the initial wet weight of the washed epithelium or tumor prior to cell isolation [22]. The yield of isolated islets is evaluated with a light microscope after dithizone (DTZ) staining. DTZ is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. Determination of the islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. Therefore, the common microscopic analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 µm. However, islets are of irregular form, which makes this determination user-dependent, and the analysis is irreproducible once the original sample is discarded. Recently, Lembert et al., showed that areal–density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single-step analysis without computer programming and is valuable for online determination of islet yield preceding transplantation [47]. An improved method of islet volume determination using digital image analysis (DIA) has been also developed to remove operator bias and automate the islet counting process. It was found that volumes determined by DIA correlated more closely with insulin content and DNA content than did conventionally determined volumes. The quantification of isolated islet tissue volume using DIA has been shown to be rapid, consistent, and objective. In the laboratory, use of this method as the standard for islet volume measurement will allow more meaningful comparison of experimental results between centers. In the clinic, its use will allow more accurate dosing of trans planted tissue [48]. The islets need to be stained with DTZ, a zinc chelating agent, which is known to selectively stain the islets of Langerhans in the pancreas.
1.44.8.2
Cell Viability
The viability of cell lots after isolation is typically assessed via trypan blue staining, in which dead cells stain blue [10, 44, 45]. The viability of cells can be confirmed using two different dyes: trypan blue exclusion and [3H]leucine uptake [22]. To assess viability, Annexin V, to identify early apoptotic cells, and 7-AAD, to identify late apoptotic and necrotic cells, may be used. One of the most
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important factors in cell transplantation is isolation of a large number of cells with good viability. Cell viability could be assessed through functional assays including static incubation and perifusion tests of glucose-stimulated insulin secretion for isolated islets. For clinical islet transplantation, viability assessment of isolated islets must be simple, rapid, sensitive, and prospective. Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation and ethidium bromide (EB) causes dead cells to fluoresce red. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDA/EB staining. The FDA/EB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation [49]. Double staining with FDA and propidium iodide (FDA/PI) is the current international standard to determine islet viability. However, a study by our group that evaluated the SYTO 13/EB (SYTO/EB) and FDA/PI techniques suggests that FDA/PI staining may overestimate islet viability and demonstrates consistently elevated values as compared to SYTO/EB. The discrepancies found between FDA/PI scoring and visual quality, when compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability [49].
1.44.8.3
Purity
Stromal cell populations are routinely assessed by phase microscopy and by immunohistochemistry using antibodies for cytokeratins and vimentin for purity. Nonmalignant and malignant cell population purity is confirmed by morphological evaluation after hematoxylin/ eosin staining and by cytokeratin immunohistochemistry (anti-Pan-Keratin clone AE1/AE3) and parallel histological analysis [22]. The purity of isolated islets is evaluated with a light microscope after DTZ staining. DTZ selectively stains pancreatic beta cells to orange. The exocrine part of the tissue remains yellow under the microscope. The purity of cells can also be analyzed by flow cytometry [8, 26, 50].
1.44.9 Conclusions Clinical outcomes of cell transplantation are influenced by numerous variables in the isolation process and pretransplant culture. Many technical challenges in these procedures must be addressed if clinical cell transplantation is to improve. Cooperation between tissue/organ transplant centers, the procurement team, and the isolation laboratory is crucial to ensure that the available cadaveric tissue/organs are referred appropriately and expediently. Cell yields remain quite variable (typically 25–75% of the potential cell mass). Moreover, clinical results vary considerably across centers in spite of comprehensive efforts to standardize isolation/ purification procedures and establish strict quality control criteria in accordance with World Health Organization (WHO) good manufacturing practice (GMP) guidelines. Production of high-quality cells is expensive, labor-intensive, and time-consuming. The process has a steep learning curve and is yet to be standardized. Despite the efforts to manufacture highly purified and standardized collagenase blends, the heterogeneity of the preparations, quality and nature of donor organs, and prolonged cold ischemia times hamper a process that is inherently difficult to control. To consistently maximize cell yield and viability for transplantation, one solution may be to optimize the cell isolation procedure. Obtaining low yield–low viability cells is mostly due to overdigestion of the tissue. It is required to either change to less-digestive-type enzymes or decrease the enzyme concentration. However, obtaining low yield–high viability cells is mostly due to underdigestion of the tissue. It is required to increase either the enzyme concentration or the incubation time. If yield remains poor, evaluating a more digestive type enzyme and/or the addition of secondary enzyme(s) might be necessary. Based upon the enzyme(s) used, setting up the preliminary dissociation conditions similar to those of the closest available reference(s) in the literature will speed up the process of optimization. After optimizing the primary enzyme’s concentration and incubation conditions, evaluating a secondary enzyme(s) may help as well. For accurate evaluation of a particular procedure’s performance, cell yield and viability should be quantified and compared. Based upon these results, the method may be judged suitable for clinical use or reoptimized [51].
References [1] Traktuev DO, Merfeld-Clauss S, Li J, et al. (2008) A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circulation Research 102: 77–85. [2] Supp DM, Wilson-Landy K, and Boyce ST (2002) Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice. The FASEB Journal 16: 797–804. [3] Espana EM, Romano AC, Kawakita T, et al. (2003) Novel enzymatic isolation of an entire viable human limbal epithelial sheet. Investigative Ophthalmology & Visual Science 44:4275–4281. [4] Gupta S, Malhi H, Gagandeep S, and Novikoff P (1999) Liver repopulation with hepatocyte transplantation: New avenues for gene and cell therapy. The Journal of Gene Medicine 1: 386–392. [5] Ryan EA, Paty BW, Senior PA, et al. (2005) Five-year follow-up after clinical islet transplantation. Current Medical Literature – Diabetes 54: 2060–2069. [6] Gesta S, Lolmède K, Daviaud D, et al. (2003) Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression. Hormone and Metabolic
Research 35: 158–163.
[7] Kern S, Eichler H, Stoeve J, et al. (2006) Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24: 1294–1301. [8] Vandenabeele S, Hochrein H, Mavaddat N, et al. (2001) Human thymus contains 2 distinct dendritic cell populations. Blood 97: 1733–1741. [9] Sollinger HW and Young CJ (1994) Procurement of Pancreatic Islets In: Lanza RP and Chick WL (eds.) Pancreatic Islet Transplantation, p. 1. Austin, TX. R.G. Landes Co. [10] Dandri M, Burda MR, Török E, et al. (2001) Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Hepatology 33: 981–988. [11] Duanmu Z, Locke D, Smigelski J, et al. (2002) Effects of dexamethasone on aryl (SULT1A1)- and hydroxysteroid (SULT2A1)-sulfotransferase gene expression in primary cultured human hepatocytes. Drug Metabolism and Disposition 30: 997–1004.
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[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51]
The Biophysical Basis
Boutilier RG (2001) Mechanisms of cell survival in hypoxia and hypothermia. Journal of Experimental Biology 204: 3171–3181. Belzer FO and Southard JH (1988) Principles of solid-organ preservation by cold storage. Transplantation 45: 673–676. Bretschneider HJ (1980) Myocardial protection. The Journal of Thoracic and Cardiovascular Surgery 28: 295–302. de Boer J, De Meester J, Smits JM, et al. (1999) Eurotransplant randomized multicenter kidney graft preservation study comparing HTK with UW and Euro-Collins. Transplant International 12: 447–453. Hatano E, Kiuchi T, Tanaka A, et al. (1997) Hepatic preservation with histidine-tryptophan-ketoglutarate solution in living-related and cadaveric liver transplantation. Clinical Science (London) 93: 81–88. Arnold JT, Kaufman DG, Seppälä M, and Lessey BA (2001) Endometrial stromal cells regulate epithelial cell growth in vitro: A new co-culture model. Human Reproduction 16:836–845. Bujalska IJ, Durrani OM, Abbott J, et al. (2007) Characterisation of 11beta-hydroxysteroid dehydrogenase 1 in human orbital adipose tissue: A comparison with subcutaneous and omental fat. Journal of Endocrinology 192: 279–288. Rodriguez AM, Pisani D, Dechesne CA, et al. (2005) Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. The Journal of Experimental Medicine 201: 1397–1405. Boquest AC, Shahdadfar A, Frønsdal K, et al. (2005) Isolation and transcription profiling of purified uncultured human stromal stem cells: Alteration of gene expression after in vitro cell culture. Molecular Biology of the Cell 16: 1131–1141. Fain JN, Madan AK, Hiler ML, et al. (2004) Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 145: 2273–2282. Emenaker NJ and Basson MD (2001) Short chain fatty acids differentially modulate cellular phenotype and c-myc protein levels in primary human nonmalignant and malignant colonocytes. Digestive Diseases and Sciences 46: 96–105. Dalerba P, Dylla SJ, Park IK, et al. (2007) Phenotypic characterization of human colorectal cancer stem cells. Proceedings of the National Academy of Sciences of the United States of America 104: 10158–10163. Le H, Arnold JT, McFann KK, and Blackman MR (2006) DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells. American Journal of Physiology. Endocrinology and Metabolism 290: E952–E960. Roy NS, Benraiss A, Wang S, et al. (2000) Promoter-targeted selection and isolation of neural progenitor cells from the adult human ventricular zone. Journal of Neuroscience Research 59: 321–331. Kanai T, Totsuka T, Uraushihara K, et al. (2003) Blockade of B7-H1 suppresses the development of chronic intestinal inflammation. Journal of Immunology 171: 4156–4163. Takano M, Meneshian A, Sheikh E, et al. (2002) Rapid up regulation of endothelial P-selectin expression via reactive oxygen species generation. American Journal of Physiology. Heart and Circulatory Physiology 283: H2054–H2061. Li W, Sabater AL, Chen YT, et al. (2007) A novel method of isolation, preservation, and expansion of human corneal endothelial cells. Investigative Ophthalmology & Visual Science 48: 614–620. Liu R, Wang X, Chen GY, et al. (2007) The prognostic role of a gene signature from tumorigenic breast-cancer cells. The New England Journal of Medicine 356: 217–226. Makrilia N, Kollias A, Manolopoulos L, and Syrigos K (2009) Cell adhesion molecules: Role and clinical significance in cancer. Cancer Investigation 27: 1023–1037. Gonzalez-Mariscal L, Contreras RG, Bolívar JJ, et al. (1990) Role of calcium in tight junction formation between epithelial cells. American Journal of Physiology 259: C978–C986. Hugenschmidt S, Planas-Bohne F, and Taylor DM (1993) On the toxicity of low doses of tetrasodium-ethylenediamine-tetraacetate (Na-EDTA) in normal rat kidney (NRK) cells in culture. Archives of Toxicology 67: 76–78. Barnett MJ, Zhai X, LeGatt DF, et al. (2005) Quantitative assessment of collagenase blends for human islet isolation. Transplantation 80: 723–728. Jeon ES, Song HY, Kim MR, et al. (2006) Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK. The Journal of Lipid Research 47: 653–664. Planat-Benard V, Silvestre JS, Cousin B, et al. (2004) Plasticity of human adipose lineage cells toward endothelial cells: Physiological and therapeutic perspectives. Circulation 109: 656–663. Fahlgren A, Hammarström S, Danielsson A, and Hammarström ML (2003) Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis. Clinical and Experimental Immunology 131: 90–101. Malhi H, Irani AN, Gagandeep S, and Gupta S (2002) Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. Journal of Cell Science 115: 2679–2688. Li A, Pouliot N, Redvers R, and Kaur P (2004) Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny. The Journal of Clinical Investigation 113: 390–400. Yu H, Fang D, Kumar SM, et al. (2006) Isolation of a novel population of multipotent adult stem cells from human hair follicles. The American Journal of Pathology 168: 1879–1888. Liagre B, Vergne-Salle P, Corbiere C, et al. (2004) Diosgenin, a plant steroid, induces apoptosis in human rheumatoid arthritis synoviocytes with cyclooxygenase-2 overexpression. Arthritis Research & Therapy 6: R373–R383. Dunham BP and Koch RJ (1998) Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Archives of Otolaryngology – Head & Neck Surgery 124: 1325–1330. Gores PF and Sutherland DE (1993) Pancreatic islet transplantation: Is purification necessary? American Journal of Surgery 166: 538–542. Quinkler M, Sinha B, Tomlinson JW, et al. (2004) Androgen generation in adipose tissue in women with simple obesity – A site-specific role for 17beta-hydroxysteroid dehydrogenase type 5. Journal of Endocrinology 183: 331–342. Seboek D, Linscheid P, Zulewski H, et al. (2004) Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation. The Journal of Clinical Endocrinology and Metabolism 89: 4833–4839. Oberlin E, Tavian M, Blazsek I, and Péault B (2002) Blood-forming potential of vascular endothelium in the human embryo. Development 129: 4147–4157. Ricordi C (1995) The Automated Method for Islet Isolation In: Ricordi C (ed.) Methods in Cell Transplantation, pp. 99–112. Austin, TX: RG Landes. Lembert N, Wesche J, Petersen P, et al. (2003) A real density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets. Cell Transplantation 12: 33–41. Stegmann JP, O’Neil JJ, Nicholson DT, and Mullon CJ (1998) Improved assessment of isolated islet tissue volume using digital image analysis. Cell Transplantation 7: 469–478. Miyamoto M, Morimoto Y, Nozawa Y, et al. (2000) Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets. Cell Transplantation 9: 681–686. Panchision DM, Chen HL, Pistollato F, et al. (2007) Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24. Stem Cells 25: 1560–1570. Rønnov-Jessen L, Villadsen R, Edwards JC, and Petersen OW (2002) Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1 mediated conversion of fibroblasts to myofibroblasts. The American Journal of Pathology 161: 471–480.
Cell Isolation from Tissue
MR Mirbolooki, H Bozorgmanesh, C Foster III, W Kuhtrieber, and JRT Lakey, University of California at Irvine, Orange, CA, USA © 2011 Elsevier B.V. All rights reserved.
1.44.1 1.44.2 1.44.3 1.44.4 1.44.5 1.44.6 1.44.6.1 1.44.6.2 1.44.6.2.1 1.44.6.2.2 1.44.6.2.3 1.44.6.2.4 1.44.6.2.5 1.44.6.2.6 1.44.6.2.7 1.44.6.2.8 1.44.6.2.9 1.44.6.2.10 1.44.7 1.44.7.1 1.44.7.2 1.44.7.3 1.44.7.4 1.44.8 1.44.8.1 1.44.8.2 1.44.8.3 1.44.9 References
Introduction Tissue/Organ Procurement Tissue/Organ Preservation Tissue/Organ Rinsing Tissue/Organ Fragmentation Cell Dissociation Nonenzymatic Dissociation Enzymatic Dissociation Stromal cells Adipocytes Thymus and umbilical cord Breast tissue Colorectal cancer cells Epithelial cells from intestine Hepatocytes Cells from skin Hair follicles Synovia and cartilage Purification Regular Centrifugation Gradient Centrifugation Adherence Fluorescence-Activated Cell Sorting Cell Yield, Viability, and Purity Assessment Cell Yield Cell Viability Purity Conclusions
Glossary cell isolation Separation of certain cells from a tissue/ organ. cytotoxicity The degree to which something is toxic to living cells. endonuclease An enzyme that cleaves the phosphodiester bond within a polynucleotide chain. lyophilized Dried by freezing in a high vacuum.
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organ perfusion Injection of a fluid into a blood vessel of the procured organ or tissues. organ preservation Transporting of donor organs, after surgical removal, for processing and transplant. organ procurement Obtaining organs for transplantation or cell isolation. reconstitution To bring back a liquid in a concentrated or powder form to its normal strength by adding water.
1.44.1 Introduction Over the past decades, interest has grown in identifying and characterizing cells isolated from a range of types of human tissues and organs. Experimental models and clinical trials have provided great opportunities to investigate the remarkable biological and clinical properties of these cells. For instance, stromal cells [1] found in many adult tissues have been an attractive stem cell source for the regeneration of damaged tissues in clinical applications; cultured skin substitutes (CSS) [2] composed of dermal fibroblasts and epidermal keratinocytes have been used as an adjunctive therapy in the treatment of large burn wounds; and transplantation of limbal tissue has replenished the stem cells population to support the regeneration of the entire corneal surface epithelium [3]. Isolation of cells from organs such as liver and pancreas has been in the center of interest in many research laboratories. The
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engraftment of transplanted hepatocytes in the liver has drawn significant interest from the field of cell therapy [4], and islet transplantation has shown the potential to prevent chronic pancreas transplantation complications in diabetics, while providing physiologic glucose control [5]. In spite of the advances in the field, there is a confusing inconsistency in the methods of cell isolation and they seem to be far from optimum. Therefore, the choice of one technique over another has often been arbitrary and based more on individual experiences rather than on an understanding of why a certain method works and what modifications could lead it to an even better outcome. The main goal of a cell isolation procedure is to optimize the yield of functionally viable, isolated cells. Many factors affect cell isolation, as it is a complex procedure. Type of tissue, donor’s factors such as body mass index (BMI) and age, warm/cold ischemia during organ procurement/preservation, isolation solution, enzyme(s), variances in any enzyme preparation, concentra tion(s) of enzyme(s), and digestion time are among the factors that affect the cell isolation outcome. Scientists searching the literature for the protocols for using digestive enzymes and optimal conditions for tissue/cell isolation are often confronted with conflicting data. As a first step, it is important to develop a consistent and reproducible method of isolating intact and viable cells. This article summarizes different tissue/cell isolation protocols to achieve a logical approach for establishing a specific cell isolation standard operating procedure.
1.44.2 Tissue/Organ Procurement Many factors limit the success of a cell isolation procedure. Donor factors such as cause of death [6] and donor age [7], along with the surgical procurement procedure, are among the key factors to be considered at the time of tissue/organ acceptance for the purpose of cell isolation and transplantation. If perfusion is not required to obtain the organ/tissue, the procedure is relatively simple and easy. Tissues are procured either from cadavers or from live donors who are undergoing surgery for another reason. Human subcutaneous adipose tissue can be obtained from healthy women undergoing abdominal dermolipectomy for plastic surgery [6]. Bone marrow aspirates can be obtained from the femoral shaft of patients undergoing total hip replacement or puncturing the iliac crest at an orthopedic department [7]. Even discarded tissue like human thymus can be obtained from young children undergoing corrective cardiac surgery [8]. However, if perfusion is required to obtain an organ like liver or pancreas, the procedure is more complicated and many factors including warm/cold ischemia could affect the outcome of cell isolation. The human pancreas is one of the most challenging organs to procure for vascularized clinical transplantation, based on a need to preserve the integrity of its capsule and of the vascular pedicles for implantation while avoiding injury to the inflow to the liver [9]. Different challenges persist when the pancreas is procured for the purpose of islet isolation, because precise procurement techniques are essential for successful isolation of large numbers of viable islets. Surgical expertise, procurement technique, and minimal ischemia time have a major impact on the recovery of functionally viable islets and posttransplant clinical outcomes. In these cases, the donor organs are typically perfused in situ with cold University of Wisconsin (UW) solution before explantation. Cold ischemia time during procurement is defined as the time after clamping of aorta until excision of the organ, which varies from 20 min to 4 h. In some cases, the organ is placed on ice and perfused with ice-cold UW solution by means of multiple catheters inserted into the vessels on the cut surface of the resected fragment immediately after excision [10]. Although the most common perfusion solution is UW in multiple organ procurement, perfusion can be done with other solutions including the simple option of Hanks’ balanced salt solution (HBSS) lacking calcium and magnesium and containing 10 mM HEPES and 5 mM ethyleneglycoltetraacetic acid (EGTA) [11].
1.44.3 Tissue/Organ Preservation During organ procurement, blood supply and hence oxygen supply is necessarily interrupted. Therefore, cells cannot continue to meet the energy demands of the active ion-transporting systems and this leads to cell death [12]. Research efforts by one of the pioneers of organ preservation Folkert O. Belzer and his colleague James H. Southard resulted in the development of a preservation solution in the late 1980s based on five philosophies [13]. They developed a UW solution containing impermeants (raffinose, lactobionate) to minimize hypothermia-induced cell swelling, buffers (phosphate) to prevent intracellular acidosis, a colloid (hydroxyethyl starch) to prevent the expansion of interstitial space during the flush-out period, free radical inhibitors and scavengers (glutathione, allopurinol) to prevent injury from oxygen free radicals during ischemia and after reperfusion, and energy precursors (Mg+, adenosine) for energy metabolism during reperfusion period. Histidine–tryptophane–ketoglutarate (HTK) solution is another common preservation solution. It was developed in the 1970s by Bretschneider as a cardioplegia solution [14] and is being used increasingly for both kidney [15] and liver [16] transplantation. HTK contains less potassium and sodium and a strong histidine buffer that increases the osmotic effect of mannitol, which is also included in this solution. Tryptophan is added as a membrane stabilizer, and ketoglutarate is added as a metabolism substrate. These solutions are designed for long-term (12–24 h) organ preservation. For shorter preservation, the media can be simple. Endometrial tissue, for instance, is transported to the laboratory in an isolation medium consisting of Dulbecco’s modified Eagle medium, high glucose (DMEM-H) culture medium and 5% fetal bovine serum (FBS) plus antibiotics [17].
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1.44.4 Tissue/Organ Rinsing The tissue pieces may need to be washed by an appropriate buffer like phosphate buffered saline (PBS) [7] or Hank’s buffered saline solution (HBSS) [18, 19] to remove contaminating debris and red blood cells. The washing solution may also contain antibiotics [18, 19]. For some tissues, 2.5 µg/ml amphotericin B [20] is added to cover a broad range of bacteria. The washing step usually takes 5–10 min; however, tissue may be incubated in buffer for a longer time (~30 min) to more efficiently reduce contamination of the tissue with blood cells and soluble factors [21]. Tissue may even be disinfected and dissociated at the same time in DMEM containing penicillin, streptomycin, and collagenase [19]. In some specific tissues like colonic mucosal speci mens, samples are thoroughly washed in PBS containing 100 IU ml−1 penicillin–streptomycin and 2 mM dithiothreitol (DTT, wash buffer) to remove debris [22]. They might also be washed in 6.5 mM DTT to remove mucus contamination. After gentle removal of the DTT solution, tissue fragments are rinsed once with HBSS [23]. The washing solution may be supplemented by 10% FBS and higher concentrations of antibiotics (200 IU ml−1) for prostate tissues [24]. For certain tissues including the brain ventricular zone [25], intestinal mucosa [26], and umbilical cord [27] tissues, washing solution is Ca/Mg-free HBSS. The corneoscleral tissue (HCECs) is rinsed three times with DMEM containing specific antibiotics (50 mg/ml gentamicin and 1.25 mg ml−1 amphotericin B) [28].
1.44.5 Tissue/Organ Fragmentation Generally, the tissues need to be cut into multiple pieces with sterile scissors or scalpel [18]. The size of the pieces varies based on the tissue type and the purpose of cell isolation. Whereas adipose tissue was cut into small pieces with average weight of 20–50 mg in one study [6], the same tissue was cut into 10–20 mg pieces in another study [21]. In some cell isolation procedures like breast cancer cell isolation, the tissue is cut into small pieces, and the pieces are then minced with a blade to yield 2–3 mm3 pieces [29]. In some organs like pancreas, the tissue is fragmented into larger pieces after enzyme perfusion.
1.44.6 Cell Dissociation 1.44.6.1
Nonenzymatic Dissociation
Cells attach to surfaces and to each other by cell surface adhesion molecules. Cell adhesion molecules (CAMs) can be classified into four major families [30]: cadherins, integrins, selectins, and immunoglobulin (Ig) CAMs. The first three are calcium and/or magnesium dependent, whereas members of the last group do not require divalent ions. Removal of calcium and magnesium causes the cells to dissociate from the surface and/or from each other. Extracellular calcium is also needed for the formation of tight junctions between cells [31]. Nonenzymatic cell dissociation media do not contain divalent ions and are often supplemented with mixtures of chelators that remove additional divalent ions from the tissues. The commonly used chelators are ethylenediaminetetraacetic acid (EDTA), EGTA, and citrate. These chelators can bind calcium and magnesium from various CAMs. Chelating agents are dissolved at concentrations of 0.1–2% in Ca/Mg-free HBSS or PBS. The solutions may also contain stabilizing agents such as glycerol. Several companies market proprietary nonenzymatic dissociation solutions and claim that they are more effective as compared to the simple chelator-containing solutions. Well-known products are from Sigma, ATCC, Cellgro (Mediatech), Millipore, and Invitrogen. Nonenzymatic dissociation solutions can typically be stored for long periods of time at room temperature. Cytotoxicity of nonenzymatic solutions is highly dependent on the cell types being treated [32]. While on the one hand it is known that chelators such as EDTA can have cytotoxic effects, on the other hand, cells can be exposed for longer periods of time to such solutions as compared to trypsin without the risk of damage associated with protein overdigestion.
1.44.6.2
Enzymatic Dissociation
A major obstacle to successful cell dissociation has been the inconsistent enzymatic activity and stability of the enzyme prepara tions. Variability in enzyme blends has been considered the most important determinant of the success or failure in isolated cell yields, and this variation in potency has been observed between, and even within, enzyme lots [33]. Enzymes are typically available as lyophilized powders. They may be stored at 2–8 °C, and special care is required when opening the enzyme vials. They should not be opened in humid areas. Any vial has to be brought to room temperature before opening. Ideally, the vials should be taken from the refrigerator at least a half hour before opening, and should be left in a dessicator. Before opening the vials, it must be made sure that it is not at all cool to the touch. Once diluted with media or buffer, proteolytic enzymes can undergo autolysis; hence enzymes should be dissolved only just prior to use. Reconstituted enzymes should not be stored at 2–8 °C; if necessary, they can be aliquoted and frozen at –20 °C. Repeated freeze–thaw cycles should be avoided. All enzymes, upon reconstitution, can be sterile filtered through a 0.22-µm-pore-size membrane. As there are variations in the isolation procedures of different types of cells, we will address the differences in enzymes usage, digestion time, enzyme blockade, and collection media between various types of cells isolated from the tissues.
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1.44.6.2.1
Stromal cells
Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside. Stromal cells can be isolated from a variety of tissues, such as bone marrow, periosteum, trabecular bone, synovium, skeletal muscle, deciduous teeth, and adipose tissues. Collagenase type I (from Clostridium histolyticum) is a crude collagenase preparation that can be used for the isolation of stromal cells. The preparation contains average amounts of caseinase, clostripain, and tryptic activities. To separate the human adipose-derived stromal cell (ASC) fraction from adipocytes, Traktuev et al. digested the tissues in collagenase type I solution under agitation for 2 h at 37 °C and centrifuged at 300 g for 8 min. They resuspended the pellet in DMEM–Ham’s Nutrient Mixture F12 (DMEM–F12) containing 10% FBS, filtered it through 250-μm filters and centrifuged at 300 g for 8 min [1]. There are different versions of stromal cells isolation protocols available in the literature with minor differences. For instance, adipose tissues are treated with collagenase type I (1 mg ml−1 in HBSS with 1% bovine serum albumin (BSA)) for just 60 min at 37 ° C with intermittent shaking [34] or for 30–60 min at the same temperature with gentle agitation [7]. The pellet is centrifuged at the same g-force (300 g) but for a shorter time (5 min) [34] or at a higher g-force (400 g) and for a longer time (10 min) [20]. The activity of the collagenase is neutralized with DMEM-LG containing 10% fetal calf serum (FCS) [7]. The pellets are resuspended in a red blood cell lysis buffer (2.06 g l−1 Tris base, 7.49 g l−1 NH4Cl, pH 7.2) for 10 min at room temperature. The suspended cells are passed first through 100-µm and then through 40-µm cell sieves [20]. The prostatic stromal cells are also digested with collagenase type I (2 mg ml−1) for 2.5 h at 37 °C on a shaking rotor. The tissue digest is vigorously pipetted and epithelial clumps settled from stromal cells for 15 min without centrifugation [24].
1.44.6.2.2
Adipocytes
Adipocytes are traditionally isolated with collagenase type II at different concentrations based on the tissue’s site. It is another form of the purified collagenase enzymes prepared to contain higher clostripain activity. Collagenase type I is also used in adipocyte dissociation. The orbital preadipocyte is digested with collagenase type I (2 mg ml−1) in HBSS for 45 min at 37 °C. The samples are centrifuged at 500 g for 1 min, and the supernatant containing connective tissue debris, collagenase, and lipid is removed leaving a pellet containing preadipocytes [18]. However, it is digested in 1 mg ml−1 collagenase type II, filtered (with 150-µm nylon mesh), and centrifuged at 200 g for 35 s. In some experiments, adipocytes are digested at 37 °C in PBS containing 2% BSA and 2 mg ml−1 collagenase for 45 min and filtered through 25 µm filters [35]. In others, adipose tissue explants are digested in DMEM containing 0.5 mg ml−1 collagenase type II and 1% BSA for 30–40 min at 37 °C under constant shaking. At the end of the incubation period, the reaction is stopped by dilution with DMEM and filtered on a silk screen in order to retain undigested explants [6]. Adipose tissue may even be dissociated for just 5–10 min in DMEM containing antibiotics, 2 mg ml−1 collagenase, and 20 mg ml−1 BSA [19].
1.44.6.2.3
Thymus and umbilical cord
Collagenase type II is also employed for thymus and umbilical cord dissociations. Thymus is cut into small fragments, suspended in 10 ml RPMI 1640 (Rosewell Park Memorial Institute) medium containing 2% FCS, collagenase (1 mg ml−1, type II), and deoxyr ibonuclease (DNAse) (0.02 mg ml−1, grade II bovine pancreatic DNAse I), and then digested with intermittent agitation for 15 min at 37 °C followed by 5 min at room temperature with constant agitation. To disrupt the dendritic cell (DC)–T cell complexes, EDTA is added (to 0.01 M final) to the digest, and incubation with agitation is continued for another 5 min. The suspension is then passed through a stainless-steel sieve to remove aggregates and stromal material [8]. Human umbilical cord segments are washed and flushed with Ca/Mg-free PBS to remove clotted blood. Sixty milliliters of a 0.1% solution of collagenase type II dissolved in Ca/Mg free PBS are gently infused into the umbilical vein and incubated at 37 °C for 20 min. The collagenase–endothelial cell suspension is then allowed to drain slowly into a 50-ml tube. This tube is centrifuged for 10 min at 1000 250 g [27].
1.44.6.2.4
Breast tissue
Breast tissue is digested by collagenase type III with very different concentrations. It is lower in secondary proteolytic contaminant activities but contains typical collagenase activity. After washing with HBSS twice, minced tissue is dissociated with 200 IU ml−1 of collagenase type III in Medium 199 at 37 °C for about 2 h. During incubation, tissue is pipetted every 30 min. Dissociation is stopped by adding 5% FBS, and the cells are diluted with Medium 199 and then filtered sequentially through a sterile 100-μm nylon mesh and a 40-μm cell strainer to obtain a single cell suspension. Cells are then washed twice with HBSS and 2% heat-inactivated calf serum [29]. Breast biopsies are cut and rotated for 24 h in a serum-free medium, DMEM–F12 supplemented with 2 mM glutamine and 50 µg ml−1 gentamicin, and a high concentration of collagenase type III (900 IU ml−1). The fibroblasts are isolated by differential centrifugation of the collagenase digest and plated in DMEM–F12 in T-25 flasks 37 °C.
1.44.6.2.5
Colorectal cancer cells
To separate colorectal cancer cells, collagenease type III is also used in combination with DNase I. DNase I is an endonuclease that cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide. After gentle removal of the DTT solution, tissue fragments are rinsed once with HBSS, resuspended in serum-free RPMI medium 1640 (2 mM L-glutamine, 120 µg ml−1 penicillin, 100 µg ml−1 streptomycin, 50 µg ml−1 ceftazidime, 0.25 µg ml−1 amphotericin-B, 20 mM HEPES) with 200 IU ml−1 collage nase type III and 100 IU ml−1 DNase I, and incubated for 2 h at 37 °C to obtain enzymatic disaggregation. Cells are then resuspended by pipetting and serially filtered by using sterile gauze, and 70- and 40-µm nylon meshes [23].
Cell Isolation from Tissue
1.44.6.2.6
595
Epithelial cells from intestine
Epithelial cells are dissociated by collagenase type IV, which has low tryptic activity. This type of collagenase is usually used when the receptors need to remain integrated. The mucosal layer of intestinal specimens is cut into small pieces and treated with 0.1 mM (small intestine) or 0.2 mM (large intestine) DTT with shaking to free the cells from the tissue. The supernatant containing epithelial cells from the luminal/villous compartment and intraepithelial lymphocytes is collected. The remaining tissue pieces are treated subsequently with 72.5 IU ml−1 of collagenase type IV in heat-inactivated human AB serum >with vigorous shaking at 37 °C for 30 min. The cell suspension is then passed through a stainless-steel sieve, resulting in a cell fraction containing epithelial cells mainly from the crypt compartment, lamina propria leucocytes, and stromal cells. Cell fractions are then washed in Tris-buffered HBSS containing 0.2% human serum albumin [36].
1.44.6.2.7
Hepatocytes
Both collagenase types I and IV have been used to isolate hepatocytes. Following 30 min of initial perfusions, the warmed liver is perfused at a flow rate of 60 ml min−1 [11] or 100 ml min−1 [10] with 1.7 l of HBSS (containing calcium and magnesium) supple mented with 0.5% BSA, 0.05% collagenase (type IV), penicillin, and streptomycin. Following the collagenase perfusion, softened sections of the liver are dissected and placed into a sterile beaker and chopped with scissors, and then 500 ml of HBSS containing 0.5% BSA, 0.02% collagenase type IV [11] or 0.05% collagenase type I [10], penicillin, and streptomycin is added to the mixture. The tissue is incubated at 37 °C for 10 min with gentle shaking, and the released cells are filtered first through sterile gauze and then through a 250-µm nylon mesh and are collected into 250-ml centrifuge bottles [11]. Hepatocytes are pelleted by centrifugation at either 500 g for 5 min at 4 °C [37] or 50 g for 3 min and are washed twice with HBSS containing 0.5% BSA, penicillin, and streptomycin [11].
1.44.6.2.8
Cells from skin
Dispase, a neutral protease, has been proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis. First, the dermis and epidermis of the skin are enzymatically separated by incubation with dispase for 2.75 h. The dermal strips are placed in endothelial cell growth medium supplemented with 10% FBS and scraped with sterile angled scissors to release microvascular endothelial cells (HDMEC). The cell suspension is centrifuged and the HDMEC are inoculated in endothelial cell growth medium into flasks coated with attachment factor. At this time collagenase may be added for inducing further cell dissociation. The scraped dermal tissue strips are minced and incubated for 1 h with collagenase for fibroblast isolation [2]. The epidermal pieces are digested in trypsin–EDTA solution and the keratinocytes are inoculated into flasks containing lethally irradiated NIH 3T3 cells in a keratinocyte growth medium. Trypsin enzyme degrades protein and it is often referred to as a proteolytic enzyme. The primary basal keratinocytes can be isolated from neonatal foreskins in 4 mg ml−1 of dispase and 3 mg ml−1 of collagenase in PBS at 37 °C for 1.5 h, and the resultant cell suspension is filtered through a 70-μm cell strainer [38].
1.44.6.2.9
Hair follicles
Hair follicles are also isolated by dispase from scalp tissues (0.5–2 cm2 or less). Tissues are rinsed, trimmed to remove excess adipose tissues, cut into small pieces, and subjected to enzymatic dissociation in 12.5 mg ml−1 dispase in DMEM for 24 h at 4 °C. After treatment, the epidermis is peeled off from the dermis, and hair follicles are plucked from the dermis. Hair follicles are rinsed thoroughly with PBS to prevent contaminating epidermal or dermal cells and are examined under an inverted microscope [39].
1.44.6.2.10
Synovia and cartilage
Synovia are minced and digested with 1.5 mg ml−1 collagenase–dispase, 1 mg ml−1 hyaluronidase, and 0.15 mg ml−1 DNase I for 3–4 h at 37 °C [40]. The septal cartilage specimens are minced into 1- to 3-mm3 cubes, placed into a spinner flask, and incubated at 37 °C in a digestion medium (collagenase type II (2.00 mg ml−1), hyaluronidase (0.10 mg ml−1), and DNase I (0.15 mg ml−1) in DMEM–F12 (1:1) medium) for 18–36 h. After digestion, the dispersed cells are filtered through a 40-μm nylon cell strainer to remove any remaining undigested clumps. The cells are then suspended with PBS and centrifuged at a low speed (1000 g for 7 min) twice to remove any remaining enzymes [41].
1.44.7 Purification It is believed that there are several advantages in transplanting highly purified isolated cells, including improved engraftment, increased safety, and reduced graft immunogenicity [42]. Even in research settings, purified cells provide more consistent and reliable data. The purification of cells from tissue extract is performed in four different ways.
1.44.7.1
Regular Centrifugation
Some cells are purified by just simple centrifugation. Preadipocytes [43] are centrifuged at 90 g for 1 min, and the top adipocyte layer is removed and then centrifuged again at 90 g for 5 min. The pellet containing preadipocytes is removed, and the cells are washed with DMEM–F12. Isolated stromovascular (SV) cells are separated from adipocytes and the medium by centrifugation in 15-ml tubes very gentle for 1 min at 400 g [21] or stronger for 10 min at 600 g [35]. The SV cells are defined as those cells isolated by
596
The Biophysical Basis
collagenase digestion that do not float. After liver digestion, cell suspension is centrifuged three times in low gravitational force (50 g, 5 min) to separate hepatocytes from nonparenchymal cells [10].
1.44.7.2
Gradient Centrifugation
SV Cell suspensions (15 ml) are applied to Histopaque-1077 gradients (15 ml) in 50-ml tubes. After centrifugation (400 g, 30 min [20] or 8–10 min [17]), the cells at the gradient interface are collected, washed in HBSS, and passed through a 30-µm mesh [20]. The bone marrow aspirates are diluted 1:5 with 2 mM EDTA–PBS. The mononuclear cell (MNC) fraction is isolated by density gradient centrifugation at 435 g for 30 min at room temperature using Ficoll-Hypaque Plus solution [44, 45]. Currently, the purification of islets from exocrine tissue is performed by continuous Ficoll gradients using a refrigerated COBE 2991 cell processor [46]. The cells are recovered from the digest by centrifugation. Then the pellet is immediately resuspended in Nycodenz medium (1.068 g cm−3 and iso-osmotic with human serum), and a low-density fraction is collected after centrifugation at 1700 g for 10 min [8].
1.44.7.3
Adherence
The cellular pellet is washed with DMEM–F12 containing 15% (v/v) FCS and seeded on 48-well plates. Cells are left overnight to attach and all unattached cells (including red blood cells) are washed the following day with HBSS [44, 45]. Nonadherent cells are removed 12–18 h after initial plating by intensely washing the plates. Adipose tissue (AT)-derived fibroblastoid adherent cells are harvested at subconfluence using trypsin [7].
1.44.7.4
Fluorescence-Activated Cell Sorting
The fluorescene-activated cell sorter is a machine that can rapidly separate the cells in a suspension on the basis of size and the color of their fluorescence. This apparatus can sort as many as 300 000 cells per minute. For dendritic cell purification, the cells are then incubated for 25 min with a mixture of monoclonal antibodies (mAbs), including anti-CD3, anti-CD8, anti-CD7, anti CD15, anti-CD19, anti-CD20, and anti-glycophorin A, in EDTA–SS containing 2% human serum. After incubation, the cells coated with mAbs are removed by two cycles of sheep anti-mouse immunoglobulin-coupled magnetic beads. The first cycle is at a 3:1 and the second at a 6:1 bead-to-cell ratio. The cells are then kept overnight at 4 °C in EDTA–SS containing 10% FCS. The next morning, the cells are incubated for 25 min at 4 °C with Cy5-conjugated anti-HLA-DR and biotinylated anti-CD11b in EDTA–SS containing 2% human serum. After two washes, the cells are incubated with streptavidin–Texas Red. DC populations are then sorted by means of a FacStar Plus [8].
1.44.8 Cell Yield, Viability, and Purity Assessment 1.44.8.1
Cell Yield
Cell yield can be estimated by counting trypan blue-stained samples, using a hemocytometer [11, 37]. However, total colonocyte yield is determined as the final wet weight of the purified cell pellet as compared to the initial wet weight of the washed epithelium or tumor prior to cell isolation [22]. The yield of isolated islets is evaluated with a light microscope after dithizone (DTZ) staining. DTZ is a zinc-chelating agent known to selectively stain pancreatic beta cells because of their high zinc content. Determination of the islet mass is important for the normalization of islet experiments in the laboratory and for the precise dosing of islets for transplantation. Therefore, the common microscopic analysis is based on individual islet sizing, calculation of the frequency distribution, and conversion into islet equivalents (IEQ), which is the volume of a spherical islet with a diameter of 150 µm. However, islets are of irregular form, which makes this determination user-dependent, and the analysis is irreproducible once the original sample is discarded. Recently, Lembert et al., showed that areal–density measurements allow a rapid and reproducible estimation of IEQ without counting individual islets. It can be performed in a single-step analysis without computer programming and is valuable for online determination of islet yield preceding transplantation [47]. An improved method of islet volume determination using digital image analysis (DIA) has been also developed to remove operator bias and automate the islet counting process. It was found that volumes determined by DIA correlated more closely with insulin content and DNA content than did conventionally determined volumes. The quantification of isolated islet tissue volume using DIA has been shown to be rapid, consistent, and objective. In the laboratory, use of this method as the standard for islet volume measurement will allow more meaningful comparison of experimental results between centers. In the clinic, its use will allow more accurate dosing of trans planted tissue [48]. The islets need to be stained with DTZ, a zinc chelating agent, which is known to selectively stain the islets of Langerhans in the pancreas.
1.44.8.2
Cell Viability
The viability of cell lots after isolation is typically assessed via trypan blue staining, in which dead cells stain blue [10, 44, 45]. The viability of cells can be confirmed using two different dyes: trypan blue exclusion and [3H]leucine uptake [22]. To assess viability, Annexin V, to identify early apoptotic cells, and 7-AAD, to identify late apoptotic and necrotic cells, may be used. One of the most
Cell Isolation from Tissue
597
important factors in cell transplantation is isolation of a large number of cells with good viability. Cell viability could be assessed through functional assays including static incubation and perifusion tests of glucose-stimulated insulin secretion for isolated islets. For clinical islet transplantation, viability assessment of isolated islets must be simple, rapid, sensitive, and prospective. Fluorescein diacetate (FDA) causes live cells to fluoresce green under blue light excitation and ethidium bromide (EB) causes dead cells to fluoresce red. Discrimination of living from dead islets by insulin secretion correlated well with viability as determined by FDA/EB staining. The FDA/EB assay prospectively and easily provides a rapid, accurate, and objective measurement of the proportion of living cells and dead cells in isolated islets for clinical islet transplantation [49]. Double staining with FDA and propidium iodide (FDA/PI) is the current international standard to determine islet viability. However, a study by our group that evaluated the SYTO 13/EB (SYTO/EB) and FDA/PI techniques suggests that FDA/PI staining may overestimate islet viability and demonstrates consistently elevated values as compared to SYTO/EB. The discrepancies found between FDA/PI scoring and visual quality, when compared with alternative stains, suggests that the FDA/PI stain may not be the optimal approach to assess islet viability [49].
1.44.8.3
Purity
Stromal cell populations are routinely assessed by phase microscopy and by immunohistochemistry using antibodies for cytokeratins and vimentin for purity. Nonmalignant and malignant cell population purity is confirmed by morphological evaluation after hematoxylin/ eosin staining and by cytokeratin immunohistochemistry (anti-Pan-Keratin clone AE1/AE3) and parallel histological analysis [22]. The purity of isolated islets is evaluated with a light microscope after DTZ staining. DTZ selectively stains pancreatic beta cells to orange. The exocrine part of the tissue remains yellow under the microscope. The purity of cells can also be analyzed by flow cytometry [8, 26, 50].
1.44.9 Conclusions Clinical outcomes of cell transplantation are influenced by numerous variables in the isolation process and pretransplant culture. Many technical challenges in these procedures must be addressed if clinical cell transplantation is to improve. Cooperation between tissue/organ transplant centers, the procurement team, and the isolation laboratory is crucial to ensure that the available cadaveric tissue/organs are referred appropriately and expediently. Cell yields remain quite variable (typically 25–75% of the potential cell mass). Moreover, clinical results vary considerably across centers in spite of comprehensive efforts to standardize isolation/ purification procedures and establish strict quality control criteria in accordance with World Health Organization (WHO) good manufacturing practice (GMP) guidelines. Production of high-quality cells is expensive, labor-intensive, and time-consuming. The process has a steep learning curve and is yet to be standardized. Despite the efforts to manufacture highly purified and standardized collagenase blends, the heterogeneity of the preparations, quality and nature of donor organs, and prolonged cold ischemia times hamper a process that is inherently difficult to control. To consistently maximize cell yield and viability for transplantation, one solution may be to optimize the cell isolation procedure. Obtaining low yield–low viability cells is mostly due to overdigestion of the tissue. It is required to either change to less-digestive-type enzymes or decrease the enzyme concentration. However, obtaining low yield–high viability cells is mostly due to underdigestion of the tissue. It is required to increase either the enzyme concentration or the incubation time. If yield remains poor, evaluating a more digestive type enzyme and/or the addition of secondary enzyme(s) might be necessary. Based upon the enzyme(s) used, setting up the preliminary dissociation conditions similar to those of the closest available reference(s) in the literature will speed up the process of optimization. After optimizing the primary enzyme’s concentration and incubation conditions, evaluating a secondary enzyme(s) may help as well. For accurate evaluation of a particular procedure’s performance, cell yield and viability should be quantified and compared. Based upon these results, the method may be judged suitable for clinical use or reoptimized [51].
References [1] Traktuev DO, Merfeld-Clauss S, Li J, et al. (2008) A population of multipotent CD34-positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circulation Research 102: 77–85. [2] Supp DM, Wilson-Landy K, and Boyce ST (2002) Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice. The FASEB Journal 16: 797–804. [3] Espana EM, Romano AC, Kawakita T, et al. (2003) Novel enzymatic isolation of an entire viable human limbal epithelial sheet. Investigative Ophthalmology & Visual Science 44:4275–4281. [4] Gupta S, Malhi H, Gagandeep S, and Novikoff P (1999) Liver repopulation with hepatocyte transplantation: New avenues for gene and cell therapy. The Journal of Gene Medicine 1: 386–392. [5] Ryan EA, Paty BW, Senior PA, et al. (2005) Five-year follow-up after clinical islet transplantation. Current Medical Literature – Diabetes 54: 2060–2069. [6] Gesta S, Lolmède K, Daviaud D, et al. (2003) Culture of human adipose tissue explants leads to profound alteration of adipocyte gene expression. Hormone and Metabolic
Research 35: 158–163.
[7] Kern S, Eichler H, Stoeve J, et al. (2006) Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24: 1294–1301. [8] Vandenabeele S, Hochrein H, Mavaddat N, et al. (2001) Human thymus contains 2 distinct dendritic cell populations. Blood 97: 1733–1741. [9] Sollinger HW and Young CJ (1994) Procurement of Pancreatic Islets In: Lanza RP and Chick WL (eds.) Pancreatic Islet Transplantation, p. 1. Austin, TX. R.G. Landes Co. [10] Dandri M, Burda MR, Török E, et al. (2001) Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus. Hepatology 33: 981–988. [11] Duanmu Z, Locke D, Smigelski J, et al. (2002) Effects of dexamethasone on aryl (SULT1A1)- and hydroxysteroid (SULT2A1)-sulfotransferase gene expression in primary cultured human hepatocytes. Drug Metabolism and Disposition 30: 997–1004.
598
[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51]
The Biophysical Basis
Boutilier RG (2001) Mechanisms of cell survival in hypoxia and hypothermia. Journal of Experimental Biology 204: 3171–3181. Belzer FO and Southard JH (1988) Principles of solid-organ preservation by cold storage. Transplantation 45: 673–676. Bretschneider HJ (1980) Myocardial protection. The Journal of Thoracic and Cardiovascular Surgery 28: 295–302. de Boer J, De Meester J, Smits JM, et al. (1999) Eurotransplant randomized multicenter kidney graft preservation study comparing HTK with UW and Euro-Collins. Transplant International 12: 447–453. Hatano E, Kiuchi T, Tanaka A, et al. (1997) Hepatic preservation with histidine-tryptophan-ketoglutarate solution in living-related and cadaveric liver transplantation. Clinical Science (London) 93: 81–88. Arnold JT, Kaufman DG, Seppälä M, and Lessey BA (2001) Endometrial stromal cells regulate epithelial cell growth in vitro: A new co-culture model. Human Reproduction 16:836–845. Bujalska IJ, Durrani OM, Abbott J, et al. (2007) Characterisation of 11beta-hydroxysteroid dehydrogenase 1 in human orbital adipose tissue: A comparison with subcutaneous and omental fat. Journal of Endocrinology 192: 279–288. Rodriguez AM, Pisani D, Dechesne CA, et al. (2005) Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. The Journal of Experimental Medicine 201: 1397–1405. Boquest AC, Shahdadfar A, Frønsdal K, et al. (2005) Isolation and transcription profiling of purified uncultured human stromal stem cells: Alteration of gene expression after in vitro cell culture. Molecular Biology of the Cell 16: 1131–1141. Fain JN, Madan AK, Hiler ML, et al. (2004) Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans. Endocrinology 145: 2273–2282. Emenaker NJ and Basson MD (2001) Short chain fatty acids differentially modulate cellular phenotype and c-myc protein levels in primary human nonmalignant and malignant colonocytes. Digestive Diseases and Sciences 46: 96–105. Dalerba P, Dylla SJ, Park IK, et al. (2007) Phenotypic characterization of human colorectal cancer stem cells. Proceedings of the National Academy of Sciences of the United States of America 104: 10158–10163. Le H, Arnold JT, McFann KK, and Blackman MR (2006) DHT and testosterone, but not DHEA or E2, differentially modulate IGF-I, IGFBP-2, and IGFBP-3 in human prostatic stromal cells. American Journal of Physiology. Endocrinology and Metabolism 290: E952–E960. Roy NS, Benraiss A, Wang S, et al. (2000) Promoter-targeted selection and isolation of neural progenitor cells from the adult human ventricular zone. Journal of Neuroscience Research 59: 321–331. Kanai T, Totsuka T, Uraushihara K, et al. (2003) Blockade of B7-H1 suppresses the development of chronic intestinal inflammation. Journal of Immunology 171: 4156–4163. Takano M, Meneshian A, Sheikh E, et al. (2002) Rapid up regulation of endothelial P-selectin expression via reactive oxygen species generation. American Journal of Physiology. Heart and Circulatory Physiology 283: H2054–H2061. Li W, Sabater AL, Chen YT, et al. (2007) A novel method of isolation, preservation, and expansion of human corneal endothelial cells. Investigative Ophthalmology & Visual Science 48: 614–620. Liu R, Wang X, Chen GY, et al. (2007) The prognostic role of a gene signature from tumorigenic breast-cancer cells. The New England Journal of Medicine 356: 217–226. Makrilia N, Kollias A, Manolopoulos L, and Syrigos K (2009) Cell adhesion molecules: Role and clinical significance in cancer. Cancer Investigation 27: 1023–1037. Gonzalez-Mariscal L, Contreras RG, Bolívar JJ, et al. (1990) Role of calcium in tight junction formation between epithelial cells. American Journal of Physiology 259: C978–C986. Hugenschmidt S, Planas-Bohne F, and Taylor DM (1993) On the toxicity of low doses of tetrasodium-ethylenediamine-tetraacetate (Na-EDTA) in normal rat kidney (NRK) cells in culture. Archives of Toxicology 67: 76–78. Barnett MJ, Zhai X, LeGatt DF, et al. (2005) Quantitative assessment of collagenase blends for human islet isolation. Transplantation 80: 723–728. Jeon ES, Song HY, Kim MR, et al. (2006) Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK. The Journal of Lipid Research 47: 653–664. Planat-Benard V, Silvestre JS, Cousin B, et al. (2004) Plasticity of human adipose lineage cells toward endothelial cells: Physiological and therapeutic perspectives. Circulation 109: 656–663. Fahlgren A, Hammarström S, Danielsson A, and Hammarström ML (2003) Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis. Clinical and Experimental Immunology 131: 90–101. Malhi H, Irani AN, Gagandeep S, and Gupta S (2002) Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. Journal of Cell Science 115: 2679–2688. Li A, Pouliot N, Redvers R, and Kaur P (2004) Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny. The Journal of Clinical Investigation 113: 390–400. Yu H, Fang D, Kumar SM, et al. (2006) Isolation of a novel population of multipotent adult stem cells from human hair follicles. The American Journal of Pathology 168: 1879–1888. Liagre B, Vergne-Salle P, Corbiere C, et al. (2004) Diosgenin, a plant steroid, induces apoptosis in human rheumatoid arthritis synoviocytes with cyclooxygenase-2 overexpression. Arthritis Research & Therapy 6: R373–R383. Dunham BP and Koch RJ (1998) Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment. Archives of Otolaryngology – Head & Neck Surgery 124: 1325–1330. Gores PF and Sutherland DE (1993) Pancreatic islet transplantation: Is purification necessary? American Journal of Surgery 166: 538–542. Quinkler M, Sinha B, Tomlinson JW, et al. (2004) Androgen generation in adipose tissue in women with simple obesity – A site-specific role for 17beta-hydroxysteroid dehydrogenase type 5. Journal of Endocrinology 183: 331–342. Seboek D, Linscheid P, Zulewski H, et al. (2004) Somatostatin is expressed and secreted by human adipose tissue upon infection and inflammation. The Journal of Clinical Endocrinology and Metabolism 89: 4833–4839. Oberlin E, Tavian M, Blazsek I, and Péault B (2002) Blood-forming potential of vascular endothelium in the human embryo. Development 129: 4147–4157. Ricordi C (1995) The Automated Method for Islet Isolation In: Ricordi C (ed.) Methods in Cell Transplantation, pp. 99–112. Austin, TX: RG Landes. Lembert N, Wesche J, Petersen P, et al. (2003) A real density measurement is a convenient method for the determination of porcine islet equivalents without counting and sizing individual islets. Cell Transplantation 12: 33–41. Stegmann JP, O’Neil JJ, Nicholson DT, and Mullon CJ (1998) Improved assessment of isolated islet tissue volume using digital image analysis. Cell Transplantation 7: 469–478. Miyamoto M, Morimoto Y, Nozawa Y, et al. (2000) Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets. Cell Transplantation 9: 681–686. Panchision DM, Chen HL, Pistollato F, et al. (2007) Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24. Stem Cells 25: 1560–1570. Rønnov-Jessen L, Villadsen R, Edwards JC, and Petersen OW (2002) Differential expression of a chloride intracellular channel gene, CLIC4, in transforming growth factor-beta1 mediated conversion of fibroblasts to myofibroblasts. The American Journal of Pathology 161: 471–480.