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Magnetic manipulation device for the optimization of cell processing conditions
Journal of Bioscience and Bioengineering VOL. 109 No. 2, 182 – 188, 2010 www.elsevier.com/locate/jbiosc
Magnetic manipulation device for the optimization of cell processing conditions Hiroshi Ito,1 Ryuji Kato,1 Kosuke Ino,2 and Hiroyuki Honda1,3,⁎ Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan 1 Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-604, Aoba, Sendai 980-8579, Japan 2 and MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan 3 Received 2 April 2009; accepted 15 July 2009 Available online 12 August 2009
Variability in human cell phenotypes make it's advancements in optimized cell processing necessary for personalized cell therapy. Here we propose a strategy of palm-top sized device to assist physically manipulating cells for optimizing cell preparations. For the design of such a device, we combined two conventional approaches: multi-well plate formatting and magnetic cell handling using magnetite cationic liposomes (MCLs). From our previous works, we showed the labeling applications of MCL on adhesive cells for various tissue engineering approaches. To feasibly transfer cells in multi-well plate, we here evaluated the magnetic response of MCL-labeled suspension type cells. The cell handling performance of Jurkat cells proved to be faster and more robust compared to MACS (Magnetic Cell Sorting) bead methods. To further confirm our strategy, prototype palm-top sized device “magnetic manipulation device (MMD)” was designed. In the device, the actual cell transportation efficacy of Jurkat cells was satisfying. Moreover, as a model of the most distributed clinical cell processing, primary peripheral blood mononuclear cells (PBMCs) from different volunteers were evaluated. By MMD, individual PBMCs indicated to have optimum Interleukin-2 (IL-2) concentrations for the expansion. Such huge differences of individual cells indicated that MMD, our proposing efficient and self-contained support tool, could assist the feasible and cost-effective optimization of cell processing in clinical facilities. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cell therapy; T cell activation; Peripheral blood mononuclear cells; Magnetic cell manipulation; Magnetic beads]
Cell-based therapy is a growing research field in regenerative medicine (1), which aims to resolve defects by using a patient's own cells. Cell therapy applications using various types of stem cells have been reported. For example, mesenchymal stem cells (MSCs) (2), hematopoietic stem cells (HSCs) (3), embryonic stem cells (ES cells) (4), and induced pluripotent stem cells (iPS cells) (5) have been studied widely. Moreover, adoptive immunotherapy using a patient's own immunocytes has been widely studied as an approach for cancer therapy. Adoptive immunotherapy has been described using various type of activated lymphocytes such as cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells (LAKs) and CD3-activated T lymphocytes (CATs) (6,7). CAT therapy using peripheral blood T lymphocytes activated with anti-CD3 Abbreviations: BSA, bovine serum albumin; CAT, CD3-activated T lymphocytes; CD3, cluster of differentiation 3; CTL, cytotoxic T lymphocytes; DLPC, dilauroyl phosphatidylcholine; DOPE, dioleoyl phosphatidylethanolamine; ES cell, embryonic stem cells; FBS, fetal bovine serum; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; HSC, hematopoietic stem cells; IL-2, interleukin-2; iPS cell, induced pluripotent stem cells; LAK, lymphokine-activated killer cells; MACS, Magnetic Cell Sorting; MCL, magnetite cationic liposomes; MMD, magnetic manipulation device; MSC, mesenchymal stem cells; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; TIL, tumor-infiltrating lymphocytes; TMAG, N-(α-trimethylammonioacetyl)didodecyl-D-glutamate chloride; Treg, regulatory T cells; WBC, white blood cell. ⁎ Corresponding author. Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Tel.: +81 052 789 3215; fax: +81 052 789 3214. E-mail address: [email protected] (H. Honda).
antibody and IL-2 is a simple cell therapy which has been adopted by many clinics as an effective support treatment for cancer patients (8). Cell-based immunotherapy is also being used to explore molecular mechanisms of autoimmune diseases, such as the role of regulatory T cells (Treg) (9). Advances in cell therapy have caused an increased demand for cell processing technology that can readily adapt to differences among individual patients. Optimizing processing conditions for each patient requires a large amount of resources. Therefore robust, low cost, automated tools that can assist with the optimization process will be required for continued growth of cell therapy applications in the clinic. In this aspect, miniaturization of automated cell-based assay systems has significant advances (10). Currently, there are miniaturized systems for cell culture (11), cell manipulation (12), cell sorting (13), and cell lysis (14). However, miniaturization of assay to micro- or nano-scales has both advantages and disadvantages. In spite of the great reduction of consumables in micro-scale cell assay (15), such tools are known to be extremely sensitive to operative errors; therefore require expensive, specialized equipment, which are often incompatible with standard equipment found in ordinary laboratories. Therefore, in the present study, we propose a strategy of palmtop sized device in cell processing optimization, which is robust in the cell assays and feasible in handling without any specialized instruments, for the assisting tool. In the proposed strategy of palm-top sized device, which we term “magnetic manipulation device (MMD)”, we combined two
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conventional technologies: magnetic cell handling and a multi-well plate format. Although cells from a patient are precious and limiting in cell therapy, the cell culture process should be investigated with many parameters (medium condition, cytokine concentration, and cell seeding density) to prepare enough number of cells for treatment. The palm-top size of the device is still effective in this aspect as microscale devices for the reduction of assay volume. For the determination of optimized cell culture condition for individuals, the multi-well plate format provides flexibility to assay various conditions simultaneously. However, liquid handling for performing cell stimulation with different soluble factors is still a laborious task. Therefore, our strategy of MMD employs a magnetic cell handling technique to provide feasible cell handling in multi-well plate format. Cell labeling and manipulation by non-toxic magnetic particles allow designing an enclosed device that assures sterile conditions for handling in various places in cell processing facilities. For cell labeling, we used magnetite cationic liposome (MCL) technology, which introduces 10-nm magnetite nanoparticles into the target cell by electrostatic absorption of encapsulating liposome (16). In previous studies, we had applied this labeling technology for the construction of various threedimensional tissues (17–20), and also for cell patterning (21, 22). However, MCL-labeling efficacy and its magnetic handling response were never tested with suspension type cells. Therefore, as one of the clinically provided cell therapies, we chose the cell-based immunotherapy, and evaluated the magnetic response and transportation efficacy of MCL for suspension type cells in this report. According to the MCL-labeled cell response, we designed a palm-top sized compact self-contained device considering the flow effect in device wells. And finally, for the applicability of our proposed strategy, the culture condition of primary peripheral blood mononuclear cells (PBMCs) from different volunteers was examined. MATERIALS AND METHODS Preparation of magnetic cationic liposomes Magnetite nanoparticles (Fe3O4; average particle size, 10 nm; Toda Kogyo, Hiroshima, Japan) were used as the MCL cores. MCLs were prepared with the core magnetite nanoparticles and a 1:2:2 molar ratio of the following three types of lipids: N-(α-trimethylammonioacetyl)-didodecyl-Dglutamate chloride (TMAG; a cationic lipid; Sogo Pharmaceutical, Tokyo, Japan), dilauroyl phosphatidylcholine (DLPC; Sigma-Aldrich, St. Louis, USA), and dioleoyl phosphatidylethanolamine (DOPE; Avanti Polar Lipids, Alabama, USA) (16). The liposome self-organized by hydrating the lipid film and vortexing together with colloidal magnetite nanoparticles. Magnetite concentrations were measured by the potassium thiocyanate method (23). All MCL concentrations are expressed as net magnetite concentration. Cell culture and magnetic labeling Jurkat human T lymphocytes were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Jurkat cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% antibiotic mixture (100 U/ml Penicillin, 100 μg/ml Streptomycin; Invitrogen), HEPES (2-[4-(2-hydroxyethyl)-1piperazinyl] ethanesulfonic acid, Dojindo Laboratories, Kumamoto, Japan), and sodium bicarbonate (Wako Pure Chemical Industries, Osaka, Japan) at 37 °C, 5% CO2, and 95% air. Human primary peripheral blood mononuclear cells (PBMCs) were collected from heparinized venous blood obtained from three healthy volunteers (23, 26, and 32-yearold males) with permission from the ethical committee of the Nagoya University Engineering Faculty. Freshly obtained blood was purified using LymphoprepTM Tubes (Axis-Shield, Oslo, Norway) according to the manufacturer's protocol. Purified PBMCs were cultured in RPMI-1640 medium supplemented with 10 ng/ml of recombinant human IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10% FBS, 1% antibiotic mixture on anti-CD3 antibody (R&D Systems, Minneapolis, USA) coated 100 mm culture dishes at 37 °C, 5% CO2, and 95% air. After 48 h, cells were collected using cell scrapers and used in MMD assays. For magnetic labeling with MCLs, Jurkat cells and PBMCs were resuspended at a final concentration of 5 × 105 cells/ml in RPMI-1640 medium containing MCLs (100 μgmagnetite/ml) and phosphate buffered saline (PBS) (50 μg-magnetite/ml), respectively. The magnetically labeled cells were collected by centrifugation and washed three times with PBS. The purified cells were resuspended in RPMI-1640 medium and counted by the trypan blue dye-exclusion method. To compare cell labeling technologies, Jurkat cells were labeled with anti-CD3 MACS beads (Miltenyi Biotec), following the manufacturer's protocol. Briefly, cells were incubated with the MACS beads in PBS solution containing bovine serum albumin (BSA) for 15 min, and washed
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by adding the PBS solution. After centrifugation, the cells were resuspended in RPMI1640 medium and counted by the trypan blue dye-exclusion method. Estimation of cell manipulation performance Magnetically labeled cells were distributed into individual media filled wells of a 96-well plate (400 μl). MCLlabeled cells were recovered by applying magnets to the covered plates (NE160 [cylindrical, ϕ10.95 × 15 mm, 520 mT, 4.00 kg], NE139 [cylindrical, 500 mT, ϕ22.5 × 25 mm, 500 mT, 24.00 kg], or NK029 [cuboid, 510 mT, 50 × 50 × 25.4 mm, 80.00 kg]: Niroku seisakusho, Kobe, Japan, [shape-type, size, surface magnetic flux density, and magnetic attractive force]). The distance between magnets and cells was adjusted by inserting 5 mm thick plastic plates or 1 mm thick glass plates. Cells that were not collected by magnetic separation were counted by using the trypan blue method described above. Design of magnetic manipulation device The magnetic manipulation device was fabricated from an acrylic plate. A silicone rubber sheet (thickness 0.5 mm) was placed between two clear units (80 × 40 mm) to keep the flow space within a single channel and the wells on the bottom of the unit. Three wells were connected by a single flow channel. The upper unit (reusable unit, thickness 5 mm), is designed to have two fluidic connection ports (diameter 4 mm) and three screws (M6 × 1.0, pitch 18 mm). The lower unit (4 mm thick) was designed to hold a silicone sheet between two units for covering a fluidic channel (width 2 mm, depth 0.5 mm), three wells (flat bottom; upper diameter 5 mm, lower diameter 3 mm, height 3 mm, pitch length 18 mm), and a bypass channel (width 1.5 mm, depth 0.5 mm) (see also Fig. 2B). Estimation of liquid flow in MMD For the computational flow simulation, the device model was designed using VisPlus (available at http://sora.cc.nagoya-u.ac.jp/ visplus/visplusD.html) with voxels in the rectangular coordinate system. The model was simulated using the incompressible fluid analysis module in the Fujitsu α-Flow software package (Fujitsu, Tokyo, Japan). The simulation was performed using the material constants of water at 20 °C (density of 998.2 kg/m3 and viscosity of 0.001 Pa/s). Boundary condition of the inlet velocity was 8.3 mm/s, assuming that volume flow was 100 μl/s, and assuming the pressure at the outlet to be zero. The simulation was limited to the status of all open wells and channel, and excluded the effect of magnet placement. The primitive equation was the Navier–Stokes equation for threedimensional, uncompressible, viscous flow. Each simulation was performed until steady state was reached. Results were visualized using VisPlus. The ideal non-volume particles were introduced in the calculation to simulate the movement of cells. Simulations were performed at the Information Technology Center of Nagoya University. MCL-labeled cell transportation in MMD Jurkat cells (5 × 105 cells/ml) were labeled with 10 μM calcein acetoxymethyl ester (Calcein AM; Invitrogen) for 1 h. MCLs were then added directly to fluorescently labeled cells and were ready for immediate use after several washing steps. Labeled Jurkat cell suspensions (1 × 105 cells/ml, 1 ml) were loaded into the MMD filled with PBS by gravity feed. The NE160 magnet was placed under the inlet to collect cells. The magnet was then placed under the first well and that well was opened to allow transfer of cells. Cells were transferred to the first well by feeding an additional 1 ml of media into the device. The first well was then closed and the magnet was removed. PBS (1 ml) was fed into the device to wash the remaining channels. This sequence of transferring cells to the remaining wells was repeated to test the transport efficiency of the device. Transport efficiency was quantified by measuring fluorescence intensity with a fluorescence plate reader (type 374, Fluoroskan Ascent, Labsystems, Helsinki, Finland) using a 485 nm excitation and 538 nm emission filter pair. PBMC culture and measurement in MMD MCL-labeled PBMC suspensions (1 × 105 cells/ml, 2 ml) were loaded into the MMD, which was filled with PBS by gravity feed from the syringe connected to the inlet in advance. Fresh culture medium (RPMI1640 supplemented with 10 or 1 ng/ml of recombinant human IL-2) was fed into the MMD while capturing cells with the NE160 magnet. For PBMC culture, the silicone rubber sheet was removed for higher air exchange and the plate was incubated at 37 °C, 5% CO2, and 95% air2. Two MMDs were used for testing each volunteer. At days 0 and 3, phase-contrast images were recorded (Olympus, Tokyo, Japan). The software analysis package Metamorph (Molecular Devices, Downingtown, PA) was used to estimate cell numbers from recorded images. Colony numbers were counted manually.
RESULTS AND DISCUSSION Evaluation of MCL-labeled cell manipulation performance There are no reports of negative effects on MSC growth or differentiation by MCLs (20). However, the influence of MCLs on suspension cells has never been examined. We first compared the growth of the Jurkat human T lymphocyte cell line in the presence and absence of MCLs (net magnetite concentration; 200 pg/cell). Anti-CD3 MACS beads, which have been tested with Jurkat cells and showed no toxicity, were used as a positive control. The average growth rate of MCL-labeled cells (2.1-fold) was not significantly different after 48 h as compared to cells alone, without MCL labeling (2.0-fold) and as compared to anti-CD3 MACS (2.2-fold).
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FIG. 1. Cell manipulation performance using MCLs. (A) Labeling time of MCL, and the magnetic response of labeled cells using a solid cylindrical-type magnet. Magnetically labeled cells (1 × 105 cells) in a 96-well plate were recovered from the cell suspension for 1 min by placing NE139 magnet on the top cover of a 96-well plate, of which the height is 11 mm from the well bottom. The horizontal axis indicates the recovered rate from total cell suspension by magnetic manipulation. Anti-CD3 MACS beads were used for comparison. N.D. indicates not determined. (B) MCL cell handling performance. Cells labeled with no incubation period or for 5 h were recovered by applying the NE139 magnet placed on the top cover of the plate of which distance is 11 mm from the well bottom. (C) Influence of magnet–cell distance and magnet size. Cells labeled for 5 h (1 × 105 cells) were recovered within 1 min. Three magnets having coequal surface magnetic flux density were used; NE160 (cylindrical, ϕ10.95 × 15 mm, surface magnetic flux density of 520 mT), NE139 (cylindrical, 500 mT, ϕ22.5 × 25 mm, 500 mT), and NK029 (cuboid, 510 mT, 50 × 50 × 25.4 mm). Data were expressed as mean ± SD (n = 3).
Next, we tested the cell manipulation performance of MCLs. By placing a solid cylindrical-type magnet on the top cover of a 96-well plate, MCL-labeled cells could be collected rapidly from the cell suspension in the well within 1 min in spite of short labeling time (Fig. 1A). Comparing with the conventional anti-CD3 MACS procedure commonly used in small scale cell separation, several advantages of MCL labeling was revealed. First, the MACS method requires 15 min incubation for labeling (manufacturer's protocol). In MCL labeling, however, less than 1 min incubation was enough for providing sufficient magnetic label to pull up most of the labeled cells from the
J. BIOSCI. BIOENG., solution. Second, MACS labeling method is limited to be preceded in PBS solution for its effective reaction with cell-specific antibodies on MACS beads. MCL could be used for sufficient labeling in both PBS and cell culture medium, since the driving force of MCL labeling is not only the electrical affinity of its cationic surface but also the activated endocytosis combined with membrane fusion mechanism of cells. Considering the cell damage in PBS solution, MCL that can be used in culture medium should be more useful. Third, in normal MACS procedure, since the magnetic response of MACS labeled cells are low, cells are collected by the special MACS column equipped with magnet. In contrast, MCL-labeled cells could be rapidly and strongly attracted to the simple cylindrical magnets placed at a backside of the bottom plate. As shown in Fig. 1B, the cell manipulation performance was comparable for cell concentrations ranging from 1 × 104 to 1 × 106 cells using MCL. In each cell concentration, labeled cells were recovered from suspension within 1 min independent of starting cell concentration. Such advantage was considered to be important to design palm top devices, since columnar magnets and the collection process of MACS method is too complex. Based on the observed cell manipulation performance, 5 × 105 cells, 100 μg-magnetite/ml was chosen as the standard cell concentration used in subsequent MMD experiments. Magnet strength and size was also evaluated (Fig. 1C). We compared three types of magnets having approximately coequal surface magnetic flux density; NE160 (cylindrical-type, ϕ10.95 × 15 mm), NE139 (cylindrical-type, ϕ22.5 × 25 mm), and NK029 (cuboid-type, 50 × 50 × 25.4 mm). The results indicate that there is no significant difference in cell recovery rate among magnets tested when placed within 15 mm of the solution. These findings led us to the conclusion that a functional MMD could be designed with the smallest magnet (NE160) at a distance of 15 mm. These results also suggest that magnet selection should consider magnetic force at a fixed distance as well as surface magnetic flux density. In this study magnetic flux densities at a fixed distance were different even in magnets with coequal surface magnetic flux density (data not shown). Since performance of each magnet was not appreciably different within the device, we selected the NE160 magnet for its compact size and narrowed magnetic force. The design of magnetic manipulation device A schematic of the MMD is illustrated in Fig. 2. The upper unit of the MMD contains an inlet and an outlet where a syringe with the medium could be inserted. Since the MMD is fabricated with a transparent acrylic plate, medium flow or cell growth is easily visible. Fig. 2A (right) is a view of cells within an MMD well. With the MMD in a closed configuration, microscopic images could be easily obtained through the screw-type gates that separate the wells from the channels. Fig. 2B illustrates solution flow from inlet to outlet in the device. To estimate the effective micro-scale liquid flow within the MMD wells, we simulated the liquid flow in the MMD device using commercially available fluid analysis software (Fig. 2C). The simulations suggest that circulation of aqueous fluids is efficient as indicated by the flow lines. Although the simulation excluded the effect of magnet and volumes of cells, the simulation result indicated that even the flow speed by gravity feed provides sufficient strength in the MMD. The simulated flow rate in the channel was fast enough to transport cells from well to well, and was expected to be sufficient to wash the channel thoroughly. The simulated flow speed in the well was fast at the entrance, although the shape of the well provided streams that carry MCL-labeled cells to the bottom area of the well where magnet captures the cells. These result suggested that the design concept of wells in MMD were efficient to enhance the magnetic capture potential at the bottom of the well without loosing the transfer speed in the channel area. Therefore, MMD could be handled without any motor or pumps that are usually introduced in palm-top sized devices. We observed some difference of flow speeds
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FIG. 2. Palm-top sized magnetic manipulation device (MMD). (A) Image of MMD (left) and phase microscope image of the closed well with cells (right). Scale bar, 100 μm. (B) Diagrams of the MMD and flow within the device. The MMD consists of two clear acrylic layers (reusable top and disposable bottom unit) and a silicone rubber sheet (thickness 0.5 mm). The top unit had two fluidic connection ports and three screw-type gates. The bottom unit has three wells (volume about 30 μl) connected by a flow channel (depth 0.5 mm) and a bypass channel (width 1.5 mm) surrounding the wells. The silicone rubber sheet was sealed with eight screw bolts along the surrounding edges of the MMD. Solutions flowed between the sheet layer and the bottom well unit layer. When pressing the silicone rubber sheet with the screw gates on the top unit, the well could be sealed from the flow channels (volume about 30 μl). The screw gate does not disturb the overall channel flow since solutions can flow through the bypass channel. As a result, the continuous flow washes out all remaining reagents in the channel and in the other open wells. When screw gates were opened, the silicone rubber sheet would expose the well and the flow would wash the exposed well. Length unit is millimeter. Blank arrows represent liquid flow. (C) Computational flow simulation inside the MMD by the Fujitsu α-Flow. For the calculation of flow, the simulation space was limited to the status of all open wells and channel (top), and cells from inlet were simulated with ideal non-volume particles (bottom). The simulation excluded the effect of magnet placement. The image is presenting a side-view of MMD wells and channels, and solid lines and particles represent flux lines (top) and ideal non-volume particles (bottom) in the well, respectively.
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between the top area of the well and the bottom area of the well. However, from our simulation results, it was deduced that further optimization of the well shape could feasibly optimize such flow speeds in the MMD. Transportation performance of MCL-labeled cell in MMD Fig. 3A illustrates the experimental design for the MCL-cell transfer assay using the MMD. With our scheme, cells could be loaded and captured in the multi-well by magnetic force without the need for manipulation by pipetting. Isolation of individual wells allowed for washing or reagent changing in designated wells. Each of these steps was completed within the enclosed device, which is important for maintaining sterility and minimizing physical cell damage. Efficiency of each step was evaluated by measuring fluorescence of the labeled Jurkat cells (Fig. 3B). The recovery efficiency in the MMD was comparable to magnetic recovery of MCL-labeled cells in a 96well plate (Fig. 1). Our results also indicated that with the slowest flow
J. BIOSCI. BIOENG., rate (6.6 μl/s), the transportation efficacy was high; 71% from first to second well, and 94% from second to third well. Such high transportation efficacy could be provided by the MCL labeling, since the magnetic response of MCL-labeled cells are extremely strong because the magnetite is introduced not only on the surfaces of cells, but also in the cytosols. The enhancement of transportation efficacy between the first well and second well was assumed to be due to the condensation of cells in the first well that lead to form larger cell aggregates that posses higher magnetic response. The efficiency was considered to be sufficient for its usage in small devices for clinical applications. For example, when the average content of white blood cells (WBCs) is approximately 4000–9000 cells/μl and lymphocytes are approximately 25% of the WBCs, and only 200 μl of normal venous blood is required to obtain an adequate cell number (1–2 × 105 cells) for one MMD assay. Therefore, typical blood sampling (2–3 ml) could provide approximately 10 to 15 MMD assays even with a low
FIG. 3. MMD cell assay design. (A) Assay procedure in the MMD. The assay consists of five steps: 1) cell seeding and capturing using the magnet, 2) reagent feeding while capturing cells in the well, 3) washing all except the sealed well, 4) reaction, and 5) cell transportation to the next well. Blank arrows represent direction of moving cells. (B) MMD cell assay method. The fluorescent and MCL-labeled cells (1 × 105 cells) were seeded into the MMD and transferred between the wells by flow with different volume flows. The number of cells was estimated by fluorescence intensity. Data is presented as mean ± SD (n = 3).
VOL. 109, 2010 transportation efficacy of 70%. As shown in our flow speed simulation (Fig. 2C), the transportation efficacy could be further increased by optimizing the well shape. Collectively, our proposed MMD concept was considered to be evaluated to have high potential to assay small volume clinical cells on the palm top. The rapid and strong labeling performance of MCL, which enables high magnetic response with in 1 min (Fig. 1), was highly advantageous than MACS antibody-based affinity beads. The concept of aseptical cell handling in designed MMD, which enables the exchange of reaction solutions without contamination of which centrifugation poses a risk for, was confirmed by the actual high transportation efficacy. Therefore, to demonstrate the applicability of MMD, we next examined the performance of the actual cell processing examination which is clinically performed. Optimization of IL-2 concentration for PBMC processing by MMD Finally, to test the MMD for optimization of personalized cell processing, we selected the expansion process of PBMCs that is the most widely provided commercial cell therapy as a model case. In the expansion of PBMCs, the most cost-effective and critical process is the IL-2 simulation step for exponential cell growth. In clinical PBMC processing, it is known that cells have wide variations of responses to IL-2 simulation. Some cells grow rapidly with low concentration of IL2, although some cells show no effect with IL-2. Since IL-2 is one of the most expensive reagent in the process, optimization of IL-2 concentration for each patient cell provides cost reduction. Therefore, we tried to demonstrate the optimization of IL-2 concentration in the expansion process of PBMCs from healthy volunteers. PBMCs purified from venous blood were stimulated by immobilized anti-CD3 antibody for several days as the expansion procedures performed in the clinic (24). We then seeded the stimulated PBMCs
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TABLE 1. Optimization of proliferation of primary PBMCs in MMD. IL-2 conc.
Volunteer 1 Volunteer 2 Volunteer 3
10 ng/ml 1 ng/ml 10 ng/ml 1 ng/ml 10 ng/ml 1 ng/ml
Cell number Day0
Day3
3339 2103 3046 2521 3177 2876
6222 3405 4108 2225 5486 5942
Growth rate [day 3/day0] 1.9 1.6 1.3 0.9 1.7 2.1
Colony number N200 μm
N100 μm
18 8 15 8 13 9
26 33 28 27 22 51
The number of the cells was estimated by the image analysis software Metamorph and colonies were counted manually.
into the MMD and monitored the IL-2 effect on further proliferation (Fig. 4 and Table 1). Testing two IL-2 concentrations in three cell samples from three volunteers, a wide variety of growth responses were observed. These results strongly indicate that there are optimal IL-2 concentrations for stimulating PBMCs. PBMC growth from volunteer 1 was greater at 10 ng/ml IL-2 as compared to 1 ng/ml. In contrast, PBMC growth from volunteer 3 was greater at 1 ng/ml versus 10 ng/ml. Comparatively, the growth of volunteer 2 was low in both concentrations. PBMCs from volunteer 1 greatly proliferated, whereas PBMCs from volunteer 2 only formed colonies without an increase in total cell number. Such morphological changes could be clearly monitored in the MMD. The colony-forming morphology is known to be an important indicator of stimulated PBMCs. However, since colony numbers did not differ among volunteers, this was not considered a reliable indicator. Taken together, these results support the idea that morphological conditions and growth response can easily be monitored with the MMD to assist optimizing personalized cell processing conditions. In this research, we have not estimated the transportation efficacy by PBMCs
FIG. 4. Optimization of clinical PBMC expansion processing by the MMD. Phase microscope images represent PBMCs from the two most disparate volunteers ((1) and (2)) at 10 ng/ml IL-2 concentration, 0 and 3 days. Scale bar, 500 μm.
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for its heterogenicity. However, the cell transportation efficacy of MMD (Fig. 3) and the PBMC culture which consisted of IL-2 stimulation and visualized cellular activity (Fig. 4) suggests that the assay composed of multiple reactions could be performed in multi-well type MMD by transferring cells from well to well. From these results, we demonstrated the variability in optimal cell processing conditions required to treat patient populations. In response to the demand for simplified methods for optimizing processing conditions and minimizing reagents, we devised the MMD. Recently, Kirschbaum et al. reported T cell activation on a single-cell level (25). However, they used dielectrophoresis-based microfluidic devices, which demand both electronic and mechanical components. In contrast, the MMD is a palm-top sized device that features multiple, scaled-down assay features with flexible, conventional manual operation. As demands grow and become more specialized, solutions for more effective cell manipulation will be investigated and the concept of the MMD has potential to be an effective tool to improve cell processing optimization and quality of cell therapy. ACKNOWLEDGMENTS The authors thank Ichiro Takahashi and Katsuya Ishii, Ph.D. (Department of Computational Science and Engineering, Nagoya University) for technical assistance with the computational flow simulation. References 1. Chien, K. R.: Regenerative medicine and human models of human disease, Nature, 453, 302–305 (2008). 2. Barry, F. P. and Murphy, J. M.: Mesenchymal stem cells: clinical applications and biological characterization, Int. J. Biochem. Cell Biol., 36, 568–584 (2004). 3. Appelbaum, F. R.: Haematopoietic cell transplantation as immunotherapy, Nature, 411, 385–389 (2001). 4. Darabi, R., Gehlbach, K., Bachoo, R. M., Kamath, S., Osawa, M., Kamm, K. E., Kyba, M., and Perlingeiro, R. C. P.: Functional skeletal muscle regeneration from differentiating embryonic stem cells, Nat. Med., 14, 134–143 (2008). 5. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 131, 861–872 (2007). 6. June, C. H.: Adoptive T cell therapy for cancer in the clinic, J. Clin. Invest., 117, 1466–1476 (2007). 7. Zakrzewski, J. L., Suh, D., Markley, J. C., Smith, O. M., King, C., Goldberg, G. L., Jenq, R., Holland, A. M., Grubin, J., and Cabrera-Perez, J., et al.: Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors, Nat. Biotechnol., 26, 453–461 (2008). 8. Takayama, T., Sekine, T., Makuuchi, M., Yamasaki, S., Kosuge, T., Yamamoto, J., Shimada, K., Sakamoto, M., Hirohashi, S., Ohashi, Y., and Kakizoe, T.: Adoptive
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Magnetic manipulation device for the optimization of cell processing conditions Hiroshi Ito,1 Ryuji Kato,1 Kosuke Ino,2 and Hiroyuki Honda1,3,⁎ Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan 1 Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-604, Aoba, Sendai 980-8579, Japan 2 and MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan 3 Received 2 April 2009; accepted 15 July 2009 Available online 12 August 2009
Variability in human cell phenotypes make it's advancements in optimized cell processing necessary for personalized cell therapy. Here we propose a strategy of palm-top sized device to assist physically manipulating cells for optimizing cell preparations. For the design of such a device, we combined two conventional approaches: multi-well plate formatting and magnetic cell handling using magnetite cationic liposomes (MCLs). From our previous works, we showed the labeling applications of MCL on adhesive cells for various tissue engineering approaches. To feasibly transfer cells in multi-well plate, we here evaluated the magnetic response of MCL-labeled suspension type cells. The cell handling performance of Jurkat cells proved to be faster and more robust compared to MACS (Magnetic Cell Sorting) bead methods. To further confirm our strategy, prototype palm-top sized device “magnetic manipulation device (MMD)” was designed. In the device, the actual cell transportation efficacy of Jurkat cells was satisfying. Moreover, as a model of the most distributed clinical cell processing, primary peripheral blood mononuclear cells (PBMCs) from different volunteers were evaluated. By MMD, individual PBMCs indicated to have optimum Interleukin-2 (IL-2) concentrations for the expansion. Such huge differences of individual cells indicated that MMD, our proposing efficient and self-contained support tool, could assist the feasible and cost-effective optimization of cell processing in clinical facilities. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Cell therapy; T cell activation; Peripheral blood mononuclear cells; Magnetic cell manipulation; Magnetic beads]
Cell-based therapy is a growing research field in regenerative medicine (1), which aims to resolve defects by using a patient's own cells. Cell therapy applications using various types of stem cells have been reported. For example, mesenchymal stem cells (MSCs) (2), hematopoietic stem cells (HSCs) (3), embryonic stem cells (ES cells) (4), and induced pluripotent stem cells (iPS cells) (5) have been studied widely. Moreover, adoptive immunotherapy using a patient's own immunocytes has been widely studied as an approach for cancer therapy. Adoptive immunotherapy has been described using various type of activated lymphocytes such as cytotoxic T lymphocytes (CTLs), tumor-infiltrating lymphocytes (TILs), lymphokine-activated killer cells (LAKs) and CD3-activated T lymphocytes (CATs) (6,7). CAT therapy using peripheral blood T lymphocytes activated with anti-CD3 Abbreviations: BSA, bovine serum albumin; CAT, CD3-activated T lymphocytes; CD3, cluster of differentiation 3; CTL, cytotoxic T lymphocytes; DLPC, dilauroyl phosphatidylcholine; DOPE, dioleoyl phosphatidylethanolamine; ES cell, embryonic stem cells; FBS, fetal bovine serum; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; HSC, hematopoietic stem cells; IL-2, interleukin-2; iPS cell, induced pluripotent stem cells; LAK, lymphokine-activated killer cells; MACS, Magnetic Cell Sorting; MCL, magnetite cationic liposomes; MMD, magnetic manipulation device; MSC, mesenchymal stem cells; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; TIL, tumor-infiltrating lymphocytes; TMAG, N-(α-trimethylammonioacetyl)didodecyl-D-glutamate chloride; Treg, regulatory T cells; WBC, white blood cell. ⁎ Corresponding author. Department of Biotechnology, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Tel.: +81 052 789 3215; fax: +81 052 789 3214. E-mail address: [email protected] (H. Honda).
antibody and IL-2 is a simple cell therapy which has been adopted by many clinics as an effective support treatment for cancer patients (8). Cell-based immunotherapy is also being used to explore molecular mechanisms of autoimmune diseases, such as the role of regulatory T cells (Treg) (9). Advances in cell therapy have caused an increased demand for cell processing technology that can readily adapt to differences among individual patients. Optimizing processing conditions for each patient requires a large amount of resources. Therefore robust, low cost, automated tools that can assist with the optimization process will be required for continued growth of cell therapy applications in the clinic. In this aspect, miniaturization of automated cell-based assay systems has significant advances (10). Currently, there are miniaturized systems for cell culture (11), cell manipulation (12), cell sorting (13), and cell lysis (14). However, miniaturization of assay to micro- or nano-scales has both advantages and disadvantages. In spite of the great reduction of consumables in micro-scale cell assay (15), such tools are known to be extremely sensitive to operative errors; therefore require expensive, specialized equipment, which are often incompatible with standard equipment found in ordinary laboratories. Therefore, in the present study, we propose a strategy of palmtop sized device in cell processing optimization, which is robust in the cell assays and feasible in handling without any specialized instruments, for the assisting tool. In the proposed strategy of palm-top sized device, which we term “magnetic manipulation device (MMD)”, we combined two
1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.07.006
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conventional technologies: magnetic cell handling and a multi-well plate format. Although cells from a patient are precious and limiting in cell therapy, the cell culture process should be investigated with many parameters (medium condition, cytokine concentration, and cell seeding density) to prepare enough number of cells for treatment. The palm-top size of the device is still effective in this aspect as microscale devices for the reduction of assay volume. For the determination of optimized cell culture condition for individuals, the multi-well plate format provides flexibility to assay various conditions simultaneously. However, liquid handling for performing cell stimulation with different soluble factors is still a laborious task. Therefore, our strategy of MMD employs a magnetic cell handling technique to provide feasible cell handling in multi-well plate format. Cell labeling and manipulation by non-toxic magnetic particles allow designing an enclosed device that assures sterile conditions for handling in various places in cell processing facilities. For cell labeling, we used magnetite cationic liposome (MCL) technology, which introduces 10-nm magnetite nanoparticles into the target cell by electrostatic absorption of encapsulating liposome (16). In previous studies, we had applied this labeling technology for the construction of various threedimensional tissues (17–20), and also for cell patterning (21, 22). However, MCL-labeling efficacy and its magnetic handling response were never tested with suspension type cells. Therefore, as one of the clinically provided cell therapies, we chose the cell-based immunotherapy, and evaluated the magnetic response and transportation efficacy of MCL for suspension type cells in this report. According to the MCL-labeled cell response, we designed a palm-top sized compact self-contained device considering the flow effect in device wells. And finally, for the applicability of our proposed strategy, the culture condition of primary peripheral blood mononuclear cells (PBMCs) from different volunteers was examined. MATERIALS AND METHODS Preparation of magnetic cationic liposomes Magnetite nanoparticles (Fe3O4; average particle size, 10 nm; Toda Kogyo, Hiroshima, Japan) were used as the MCL cores. MCLs were prepared with the core magnetite nanoparticles and a 1:2:2 molar ratio of the following three types of lipids: N-(α-trimethylammonioacetyl)-didodecyl-Dglutamate chloride (TMAG; a cationic lipid; Sogo Pharmaceutical, Tokyo, Japan), dilauroyl phosphatidylcholine (DLPC; Sigma-Aldrich, St. Louis, USA), and dioleoyl phosphatidylethanolamine (DOPE; Avanti Polar Lipids, Alabama, USA) (16). The liposome self-organized by hydrating the lipid film and vortexing together with colloidal magnetite nanoparticles. Magnetite concentrations were measured by the potassium thiocyanate method (23). All MCL concentrations are expressed as net magnetite concentration. Cell culture and magnetic labeling Jurkat human T lymphocytes were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Jurkat cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 1% antibiotic mixture (100 U/ml Penicillin, 100 μg/ml Streptomycin; Invitrogen), HEPES (2-[4-(2-hydroxyethyl)-1piperazinyl] ethanesulfonic acid, Dojindo Laboratories, Kumamoto, Japan), and sodium bicarbonate (Wako Pure Chemical Industries, Osaka, Japan) at 37 °C, 5% CO2, and 95% air. Human primary peripheral blood mononuclear cells (PBMCs) were collected from heparinized venous blood obtained from three healthy volunteers (23, 26, and 32-yearold males) with permission from the ethical committee of the Nagoya University Engineering Faculty. Freshly obtained blood was purified using LymphoprepTM Tubes (Axis-Shield, Oslo, Norway) according to the manufacturer's protocol. Purified PBMCs were cultured in RPMI-1640 medium supplemented with 10 ng/ml of recombinant human IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10% FBS, 1% antibiotic mixture on anti-CD3 antibody (R&D Systems, Minneapolis, USA) coated 100 mm culture dishes at 37 °C, 5% CO2, and 95% air. After 48 h, cells were collected using cell scrapers and used in MMD assays. For magnetic labeling with MCLs, Jurkat cells and PBMCs were resuspended at a final concentration of 5 × 105 cells/ml in RPMI-1640 medium containing MCLs (100 μgmagnetite/ml) and phosphate buffered saline (PBS) (50 μg-magnetite/ml), respectively. The magnetically labeled cells were collected by centrifugation and washed three times with PBS. The purified cells were resuspended in RPMI-1640 medium and counted by the trypan blue dye-exclusion method. To compare cell labeling technologies, Jurkat cells were labeled with anti-CD3 MACS beads (Miltenyi Biotec), following the manufacturer's protocol. Briefly, cells were incubated with the MACS beads in PBS solution containing bovine serum albumin (BSA) for 15 min, and washed
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by adding the PBS solution. After centrifugation, the cells were resuspended in RPMI1640 medium and counted by the trypan blue dye-exclusion method. Estimation of cell manipulation performance Magnetically labeled cells were distributed into individual media filled wells of a 96-well plate (400 μl). MCLlabeled cells were recovered by applying magnets to the covered plates (NE160 [cylindrical, ϕ10.95 × 15 mm, 520 mT, 4.00 kg], NE139 [cylindrical, 500 mT, ϕ22.5 × 25 mm, 500 mT, 24.00 kg], or NK029 [cuboid, 510 mT, 50 × 50 × 25.4 mm, 80.00 kg]: Niroku seisakusho, Kobe, Japan, [shape-type, size, surface magnetic flux density, and magnetic attractive force]). The distance between magnets and cells was adjusted by inserting 5 mm thick plastic plates or 1 mm thick glass plates. Cells that were not collected by magnetic separation were counted by using the trypan blue method described above. Design of magnetic manipulation device The magnetic manipulation device was fabricated from an acrylic plate. A silicone rubber sheet (thickness 0.5 mm) was placed between two clear units (80 × 40 mm) to keep the flow space within a single channel and the wells on the bottom of the unit. Three wells were connected by a single flow channel. The upper unit (reusable unit, thickness 5 mm), is designed to have two fluidic connection ports (diameter 4 mm) and three screws (M6 × 1.0, pitch 18 mm). The lower unit (4 mm thick) was designed to hold a silicone sheet between two units for covering a fluidic channel (width 2 mm, depth 0.5 mm), three wells (flat bottom; upper diameter 5 mm, lower diameter 3 mm, height 3 mm, pitch length 18 mm), and a bypass channel (width 1.5 mm, depth 0.5 mm) (see also Fig. 2B). Estimation of liquid flow in MMD For the computational flow simulation, the device model was designed using VisPlus (available at http://sora.cc.nagoya-u.ac.jp/ visplus/visplusD.html) with voxels in the rectangular coordinate system. The model was simulated using the incompressible fluid analysis module in the Fujitsu α-Flow software package (Fujitsu, Tokyo, Japan). The simulation was performed using the material constants of water at 20 °C (density of 998.2 kg/m3 and viscosity of 0.001 Pa/s). Boundary condition of the inlet velocity was 8.3 mm/s, assuming that volume flow was 100 μl/s, and assuming the pressure at the outlet to be zero. The simulation was limited to the status of all open wells and channel, and excluded the effect of magnet placement. The primitive equation was the Navier–Stokes equation for threedimensional, uncompressible, viscous flow. Each simulation was performed until steady state was reached. Results were visualized using VisPlus. The ideal non-volume particles were introduced in the calculation to simulate the movement of cells. Simulations were performed at the Information Technology Center of Nagoya University. MCL-labeled cell transportation in MMD Jurkat cells (5 × 105 cells/ml) were labeled with 10 μM calcein acetoxymethyl ester (Calcein AM; Invitrogen) for 1 h. MCLs were then added directly to fluorescently labeled cells and were ready for immediate use after several washing steps. Labeled Jurkat cell suspensions (1 × 105 cells/ml, 1 ml) were loaded into the MMD filled with PBS by gravity feed. The NE160 magnet was placed under the inlet to collect cells. The magnet was then placed under the first well and that well was opened to allow transfer of cells. Cells were transferred to the first well by feeding an additional 1 ml of media into the device. The first well was then closed and the magnet was removed. PBS (1 ml) was fed into the device to wash the remaining channels. This sequence of transferring cells to the remaining wells was repeated to test the transport efficiency of the device. Transport efficiency was quantified by measuring fluorescence intensity with a fluorescence plate reader (type 374, Fluoroskan Ascent, Labsystems, Helsinki, Finland) using a 485 nm excitation and 538 nm emission filter pair. PBMC culture and measurement in MMD MCL-labeled PBMC suspensions (1 × 105 cells/ml, 2 ml) were loaded into the MMD, which was filled with PBS by gravity feed from the syringe connected to the inlet in advance. Fresh culture medium (RPMI1640 supplemented with 10 or 1 ng/ml of recombinant human IL-2) was fed into the MMD while capturing cells with the NE160 magnet. For PBMC culture, the silicone rubber sheet was removed for higher air exchange and the plate was incubated at 37 °C, 5% CO2, and 95% air2. Two MMDs were used for testing each volunteer. At days 0 and 3, phase-contrast images were recorded (Olympus, Tokyo, Japan). The software analysis package Metamorph (Molecular Devices, Downingtown, PA) was used to estimate cell numbers from recorded images. Colony numbers were counted manually.
RESULTS AND DISCUSSION Evaluation of MCL-labeled cell manipulation performance There are no reports of negative effects on MSC growth or differentiation by MCLs (20). However, the influence of MCLs on suspension cells has never been examined. We first compared the growth of the Jurkat human T lymphocyte cell line in the presence and absence of MCLs (net magnetite concentration; 200 pg/cell). Anti-CD3 MACS beads, which have been tested with Jurkat cells and showed no toxicity, were used as a positive control. The average growth rate of MCL-labeled cells (2.1-fold) was not significantly different after 48 h as compared to cells alone, without MCL labeling (2.0-fold) and as compared to anti-CD3 MACS (2.2-fold).
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FIG. 1. Cell manipulation performance using MCLs. (A) Labeling time of MCL, and the magnetic response of labeled cells using a solid cylindrical-type magnet. Magnetically labeled cells (1 × 105 cells) in a 96-well plate were recovered from the cell suspension for 1 min by placing NE139 magnet on the top cover of a 96-well plate, of which the height is 11 mm from the well bottom. The horizontal axis indicates the recovered rate from total cell suspension by magnetic manipulation. Anti-CD3 MACS beads were used for comparison. N.D. indicates not determined. (B) MCL cell handling performance. Cells labeled with no incubation period or for 5 h were recovered by applying the NE139 magnet placed on the top cover of the plate of which distance is 11 mm from the well bottom. (C) Influence of magnet–cell distance and magnet size. Cells labeled for 5 h (1 × 105 cells) were recovered within 1 min. Three magnets having coequal surface magnetic flux density were used; NE160 (cylindrical, ϕ10.95 × 15 mm, surface magnetic flux density of 520 mT), NE139 (cylindrical, 500 mT, ϕ22.5 × 25 mm, 500 mT), and NK029 (cuboid, 510 mT, 50 × 50 × 25.4 mm). Data were expressed as mean ± SD (n = 3).
Next, we tested the cell manipulation performance of MCLs. By placing a solid cylindrical-type magnet on the top cover of a 96-well plate, MCL-labeled cells could be collected rapidly from the cell suspension in the well within 1 min in spite of short labeling time (Fig. 1A). Comparing with the conventional anti-CD3 MACS procedure commonly used in small scale cell separation, several advantages of MCL labeling was revealed. First, the MACS method requires 15 min incubation for labeling (manufacturer's protocol). In MCL labeling, however, less than 1 min incubation was enough for providing sufficient magnetic label to pull up most of the labeled cells from the
J. BIOSCI. BIOENG., solution. Second, MACS labeling method is limited to be preceded in PBS solution for its effective reaction with cell-specific antibodies on MACS beads. MCL could be used for sufficient labeling in both PBS and cell culture medium, since the driving force of MCL labeling is not only the electrical affinity of its cationic surface but also the activated endocytosis combined with membrane fusion mechanism of cells. Considering the cell damage in PBS solution, MCL that can be used in culture medium should be more useful. Third, in normal MACS procedure, since the magnetic response of MACS labeled cells are low, cells are collected by the special MACS column equipped with magnet. In contrast, MCL-labeled cells could be rapidly and strongly attracted to the simple cylindrical magnets placed at a backside of the bottom plate. As shown in Fig. 1B, the cell manipulation performance was comparable for cell concentrations ranging from 1 × 104 to 1 × 106 cells using MCL. In each cell concentration, labeled cells were recovered from suspension within 1 min independent of starting cell concentration. Such advantage was considered to be important to design palm top devices, since columnar magnets and the collection process of MACS method is too complex. Based on the observed cell manipulation performance, 5 × 105 cells, 100 μg-magnetite/ml was chosen as the standard cell concentration used in subsequent MMD experiments. Magnet strength and size was also evaluated (Fig. 1C). We compared three types of magnets having approximately coequal surface magnetic flux density; NE160 (cylindrical-type, ϕ10.95 × 15 mm), NE139 (cylindrical-type, ϕ22.5 × 25 mm), and NK029 (cuboid-type, 50 × 50 × 25.4 mm). The results indicate that there is no significant difference in cell recovery rate among magnets tested when placed within 15 mm of the solution. These findings led us to the conclusion that a functional MMD could be designed with the smallest magnet (NE160) at a distance of 15 mm. These results also suggest that magnet selection should consider magnetic force at a fixed distance as well as surface magnetic flux density. In this study magnetic flux densities at a fixed distance were different even in magnets with coequal surface magnetic flux density (data not shown). Since performance of each magnet was not appreciably different within the device, we selected the NE160 magnet for its compact size and narrowed magnetic force. The design of magnetic manipulation device A schematic of the MMD is illustrated in Fig. 2. The upper unit of the MMD contains an inlet and an outlet where a syringe with the medium could be inserted. Since the MMD is fabricated with a transparent acrylic plate, medium flow or cell growth is easily visible. Fig. 2A (right) is a view of cells within an MMD well. With the MMD in a closed configuration, microscopic images could be easily obtained through the screw-type gates that separate the wells from the channels. Fig. 2B illustrates solution flow from inlet to outlet in the device. To estimate the effective micro-scale liquid flow within the MMD wells, we simulated the liquid flow in the MMD device using commercially available fluid analysis software (Fig. 2C). The simulations suggest that circulation of aqueous fluids is efficient as indicated by the flow lines. Although the simulation excluded the effect of magnet and volumes of cells, the simulation result indicated that even the flow speed by gravity feed provides sufficient strength in the MMD. The simulated flow rate in the channel was fast enough to transport cells from well to well, and was expected to be sufficient to wash the channel thoroughly. The simulated flow speed in the well was fast at the entrance, although the shape of the well provided streams that carry MCL-labeled cells to the bottom area of the well where magnet captures the cells. These result suggested that the design concept of wells in MMD were efficient to enhance the magnetic capture potential at the bottom of the well without loosing the transfer speed in the channel area. Therefore, MMD could be handled without any motor or pumps that are usually introduced in palm-top sized devices. We observed some difference of flow speeds
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FIG. 2. Palm-top sized magnetic manipulation device (MMD). (A) Image of MMD (left) and phase microscope image of the closed well with cells (right). Scale bar, 100 μm. (B) Diagrams of the MMD and flow within the device. The MMD consists of two clear acrylic layers (reusable top and disposable bottom unit) and a silicone rubber sheet (thickness 0.5 mm). The top unit had two fluidic connection ports and three screw-type gates. The bottom unit has three wells (volume about 30 μl) connected by a flow channel (depth 0.5 mm) and a bypass channel (width 1.5 mm) surrounding the wells. The silicone rubber sheet was sealed with eight screw bolts along the surrounding edges of the MMD. Solutions flowed between the sheet layer and the bottom well unit layer. When pressing the silicone rubber sheet with the screw gates on the top unit, the well could be sealed from the flow channels (volume about 30 μl). The screw gate does not disturb the overall channel flow since solutions can flow through the bypass channel. As a result, the continuous flow washes out all remaining reagents in the channel and in the other open wells. When screw gates were opened, the silicone rubber sheet would expose the well and the flow would wash the exposed well. Length unit is millimeter. Blank arrows represent liquid flow. (C) Computational flow simulation inside the MMD by the Fujitsu α-Flow. For the calculation of flow, the simulation space was limited to the status of all open wells and channel (top), and cells from inlet were simulated with ideal non-volume particles (bottom). The simulation excluded the effect of magnet placement. The image is presenting a side-view of MMD wells and channels, and solid lines and particles represent flux lines (top) and ideal non-volume particles (bottom) in the well, respectively.
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between the top area of the well and the bottom area of the well. However, from our simulation results, it was deduced that further optimization of the well shape could feasibly optimize such flow speeds in the MMD. Transportation performance of MCL-labeled cell in MMD Fig. 3A illustrates the experimental design for the MCL-cell transfer assay using the MMD. With our scheme, cells could be loaded and captured in the multi-well by magnetic force without the need for manipulation by pipetting. Isolation of individual wells allowed for washing or reagent changing in designated wells. Each of these steps was completed within the enclosed device, which is important for maintaining sterility and minimizing physical cell damage. Efficiency of each step was evaluated by measuring fluorescence of the labeled Jurkat cells (Fig. 3B). The recovery efficiency in the MMD was comparable to magnetic recovery of MCL-labeled cells in a 96well plate (Fig. 1). Our results also indicated that with the slowest flow
J. BIOSCI. BIOENG., rate (6.6 μl/s), the transportation efficacy was high; 71% from first to second well, and 94% from second to third well. Such high transportation efficacy could be provided by the MCL labeling, since the magnetic response of MCL-labeled cells are extremely strong because the magnetite is introduced not only on the surfaces of cells, but also in the cytosols. The enhancement of transportation efficacy between the first well and second well was assumed to be due to the condensation of cells in the first well that lead to form larger cell aggregates that posses higher magnetic response. The efficiency was considered to be sufficient for its usage in small devices for clinical applications. For example, when the average content of white blood cells (WBCs) is approximately 4000–9000 cells/μl and lymphocytes are approximately 25% of the WBCs, and only 200 μl of normal venous blood is required to obtain an adequate cell number (1–2 × 105 cells) for one MMD assay. Therefore, typical blood sampling (2–3 ml) could provide approximately 10 to 15 MMD assays even with a low
FIG. 3. MMD cell assay design. (A) Assay procedure in the MMD. The assay consists of five steps: 1) cell seeding and capturing using the magnet, 2) reagent feeding while capturing cells in the well, 3) washing all except the sealed well, 4) reaction, and 5) cell transportation to the next well. Blank arrows represent direction of moving cells. (B) MMD cell assay method. The fluorescent and MCL-labeled cells (1 × 105 cells) were seeded into the MMD and transferred between the wells by flow with different volume flows. The number of cells was estimated by fluorescence intensity. Data is presented as mean ± SD (n = 3).
VOL. 109, 2010 transportation efficacy of 70%. As shown in our flow speed simulation (Fig. 2C), the transportation efficacy could be further increased by optimizing the well shape. Collectively, our proposed MMD concept was considered to be evaluated to have high potential to assay small volume clinical cells on the palm top. The rapid and strong labeling performance of MCL, which enables high magnetic response with in 1 min (Fig. 1), was highly advantageous than MACS antibody-based affinity beads. The concept of aseptical cell handling in designed MMD, which enables the exchange of reaction solutions without contamination of which centrifugation poses a risk for, was confirmed by the actual high transportation efficacy. Therefore, to demonstrate the applicability of MMD, we next examined the performance of the actual cell processing examination which is clinically performed. Optimization of IL-2 concentration for PBMC processing by MMD Finally, to test the MMD for optimization of personalized cell processing, we selected the expansion process of PBMCs that is the most widely provided commercial cell therapy as a model case. In the expansion of PBMCs, the most cost-effective and critical process is the IL-2 simulation step for exponential cell growth. In clinical PBMC processing, it is known that cells have wide variations of responses to IL-2 simulation. Some cells grow rapidly with low concentration of IL2, although some cells show no effect with IL-2. Since IL-2 is one of the most expensive reagent in the process, optimization of IL-2 concentration for each patient cell provides cost reduction. Therefore, we tried to demonstrate the optimization of IL-2 concentration in the expansion process of PBMCs from healthy volunteers. PBMCs purified from venous blood were stimulated by immobilized anti-CD3 antibody for several days as the expansion procedures performed in the clinic (24). We then seeded the stimulated PBMCs
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TABLE 1. Optimization of proliferation of primary PBMCs in MMD. IL-2 conc.
Volunteer 1 Volunteer 2 Volunteer 3
10 ng/ml 1 ng/ml 10 ng/ml 1 ng/ml 10 ng/ml 1 ng/ml
Cell number Day0
Day3
3339 2103 3046 2521 3177 2876
6222 3405 4108 2225 5486 5942
Growth rate [day 3/day0] 1.9 1.6 1.3 0.9 1.7 2.1
Colony number N200 μm
N100 μm
18 8 15 8 13 9
26 33 28 27 22 51
The number of the cells was estimated by the image analysis software Metamorph and colonies were counted manually.
into the MMD and monitored the IL-2 effect on further proliferation (Fig. 4 and Table 1). Testing two IL-2 concentrations in three cell samples from three volunteers, a wide variety of growth responses were observed. These results strongly indicate that there are optimal IL-2 concentrations for stimulating PBMCs. PBMC growth from volunteer 1 was greater at 10 ng/ml IL-2 as compared to 1 ng/ml. In contrast, PBMC growth from volunteer 3 was greater at 1 ng/ml versus 10 ng/ml. Comparatively, the growth of volunteer 2 was low in both concentrations. PBMCs from volunteer 1 greatly proliferated, whereas PBMCs from volunteer 2 only formed colonies without an increase in total cell number. Such morphological changes could be clearly monitored in the MMD. The colony-forming morphology is known to be an important indicator of stimulated PBMCs. However, since colony numbers did not differ among volunteers, this was not considered a reliable indicator. Taken together, these results support the idea that morphological conditions and growth response can easily be monitored with the MMD to assist optimizing personalized cell processing conditions. In this research, we have not estimated the transportation efficacy by PBMCs
FIG. 4. Optimization of clinical PBMC expansion processing by the MMD. Phase microscope images represent PBMCs from the two most disparate volunteers ((1) and (2)) at 10 ng/ml IL-2 concentration, 0 and 3 days. Scale bar, 500 μm.
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for its heterogenicity. However, the cell transportation efficacy of MMD (Fig. 3) and the PBMC culture which consisted of IL-2 stimulation and visualized cellular activity (Fig. 4) suggests that the assay composed of multiple reactions could be performed in multi-well type MMD by transferring cells from well to well. From these results, we demonstrated the variability in optimal cell processing conditions required to treat patient populations. In response to the demand for simplified methods for optimizing processing conditions and minimizing reagents, we devised the MMD. Recently, Kirschbaum et al. reported T cell activation on a single-cell level (25). However, they used dielectrophoresis-based microfluidic devices, which demand both electronic and mechanical components. In contrast, the MMD is a palm-top sized device that features multiple, scaled-down assay features with flexible, conventional manual operation. As demands grow and become more specialized, solutions for more effective cell manipulation will be investigated and the concept of the MMD has potential to be an effective tool to improve cell processing optimization and quality of cell therapy. ACKNOWLEDGMENTS The authors thank Ichiro Takahashi and Katsuya Ishii, Ph.D. (Department of Computational Science and Engineering, Nagoya University) for technical assistance with the computational flow simulation. References 1. Chien, K. R.: Regenerative medicine and human models of human disease, Nature, 453, 302–305 (2008). 2. Barry, F. P. and Murphy, J. M.: Mesenchymal stem cells: clinical applications and biological characterization, Int. J. Biochem. Cell Biol., 36, 568–584 (2004). 3. Appelbaum, F. R.: Haematopoietic cell transplantation as immunotherapy, Nature, 411, 385–389 (2001). 4. Darabi, R., Gehlbach, K., Bachoo, R. M., Kamath, S., Osawa, M., Kamm, K. E., Kyba, M., and Perlingeiro, R. C. P.: Functional skeletal muscle regeneration from differentiating embryonic stem cells, Nat. Med., 14, 134–143 (2008). 5. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S.: Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell, 131, 861–872 (2007). 6. June, C. H.: Adoptive T cell therapy for cancer in the clinic, J. Clin. Invest., 117, 1466–1476 (2007). 7. Zakrzewski, J. L., Suh, D., Markley, J. C., Smith, O. M., King, C., Goldberg, G. L., Jenq, R., Holland, A. M., Grubin, J., and Cabrera-Perez, J., et al.: Tumor immunotherapy across MHC barriers using allogeneic T-cell precursors, Nat. Biotechnol., 26, 453–461 (2008). 8. Takayama, T., Sekine, T., Makuuchi, M., Yamasaki, S., Kosuge, T., Yamamoto, J., Shimada, K., Sakamoto, M., Hirohashi, S., Ohashi, Y., and Kakizoe, T.: Adoptive
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