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Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors
Acta Biomaterialia 6 (2010) 2548–2553
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Brief communication
Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors Carmela Palmiero a,c, Giorgia Imparato a,c, Francesco Urciuolo b,c, Paolo Netti a,c,* a
Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, P. Le Tecchio 80, 80125 Naples, Italy Institute of Composite and Biomedical Materials (IMCB), National Council Research (CNR), P. Le Tecchio 80, 80125 Naples, Italy c Italian Institute of Technology (IIT), Via Morego 30, 16163 Genoa, Italy b
a r t i c l e
i n f o
Article history: Received 9 October 2009 Received in revised form 29 December 2009 Accepted 19 January 2010 Available online 25 January 2010 Keywords: Tissue engineering Microcarriers Bio-fabrication Dermis
a b s t r a c t Tissue-engineered constructs can be fabricated by the assembly of smaller building blocks in order to mimic much of the native biology that is often made from repeating functional units. Our aim was to realize a three-dimensional (3-D) tissue-like construct in vitro by inducing the assembly of functional micrometric tissue precursors (lTPs). lTPs were obtained by dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. During the dynamic seeding, cells adhered, proliferated and synthesized a thin layer of extracellular matrix (ECM) in and around the macroporous beads, generating the lTPs. The analysis showed that the ECM produced was rich in type I collagen. The cells and ECM layer around the lTPs allowed their biological sintering via cell–cell and cell–matrix interaction after only a few days of dynamic seeding. The assembling ability of lTPs was exploited by placing them in a maturation chamber. After 1 week of culture disc-shaped constructs (1 cm in diameter, 1 mm in thickness) of completely assembled lTPs were collected. The biohybrid obtained presented both a homogeneous and compact aspect. Moreover, histological and immunohistochemical analyses revealed an abundant ECM, rich in type I collagen, interconnecting the lTPs. The results obtained in this survey pave the way to realizing a 3-D dermal tissue equivalent by means of a bottom-up tissue engineering approach. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Most living tissues are composed of repeating units on the scale of hundreds of microns, which are ensembles of different cell types with well defined three-dimensional (3-D) micro-architectures and tissue-specific, functional properties. To generate engineered tissues, the recreation of these structural features is of great importance in enabling the resulting function [1]. However, the classical tissue engineering approach, based on seeding cells into a preformed, porous and biodegradable scaffold to recreate the natural tissue complex structure, faces serious hurdles. Selection of the ideal biomaterial scaffold for a given cell type is problematic and has been accomplished to date mostly by trial and error. Even if the right biomaterial is available, achieving a high enough cell density and the homogeneous cell distribution necessary to construct a viable tissue is extremely time consuming. Furthermore, preshaping the scaffold may present further difficulties [2]. To overcome these limitations, recent efforts [3–11] have been concentrated on scaffold-less tissue engineering and bot-
* Corresponding author. Address: Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, P. Le Tecchio 80, 80125 Naples, Italy. Tel.: +39 081 7682408; fax: +39 081 7682404. E-mail address: [email protected] (P. Netti).
tom-up approaches aimed at generating a larger tissue construct by the assembly of smaller building blocks, which mimics the in vivo tissue structure of repeating functional units. A similar strategy was proposed by Du et al. [1], who used a bottom-up approach to direct the assembly of cell-laden microgels to generate 3D tissue with tunable microarchitecture and complexity. McGuigan and Sefton [3] proposed another interesting use of modular components for generating tissue. In their approach, rod-shaped collagen microgels that were seeded with HepG2 hepatocytes on the inside and endothelial cells on the surface were ‘‘packed” together within a bioreactor and perfused with medium. Further examples of constructs resulting from a bottom-up approach include beating cardiac sheets, generated by the stacking of sheets of cells for patches obtained by means of ‘‘cell sheet technology” [4]. In this technology, a cell sheet produced by a wellestablished 2-D cell culture system plays the role of a building block of organ-like structures. By using thermoresponsive culture dishes, cell sheets are easily harvested as intact monolayers along with their deposited extracellular matrix (ECM) and can be layered on top of one another to create a 3-D construct, such as thick cardiac muscle [12–15]. The use of cell sheets has the advantage that an entirely natural neo-tissue assembled from cells with a mature ECM can be engineered. However, this technology has a number of shortcomings,
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.01.026
C. Palmiero et al. / Acta Biomaterialia 6 (2010) 2548–2553
such as handling difficulties with the cell sheets and the limited number of cell sheets that can be effectively layered without core ischemia or hypoxia [4,16,17]. In this paper, we propose a tissue engineering strategy that allows the production of viable thick 3-D tissues with a compact ECM by using a bottom-up approach. Micrometric tissue precursors (lTPs) were obtained by means of dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. Numerous studies have illustrated that the particle cultivation technique is more effective than cell culture on flat substrates such as culture dishes [18,19]. In this study, we observed that under optimal culture conditions cells were able to adhere, proliferate and in particular synthesize ECM components to form a thin layer of tissue around the microbeads, generating microscale tissue units which we refer to as lTPs. The lTPs have been used as an ideal ‘‘material” for bio-fabrication of 3-D tissue constructs. Indeed, we demonstrate that lTPs can be assembled, in an appropriate assembling chamber, by means of the tissue layers surrounding them, which allow their fusion through cell–cell and cell–matrix interactions. Following this strategy, a 3-D functional dermal tissue equivalent has been realized. 2. Materials and methods 2.1. Sampling and characterization of lTPs 2.1.1. Cell culture and dynamic cell seeding Primary bovine dermal fibroblasts (BF-AG10385 Coriell) were propagated in monolayer culture in a 150 mm Petri dish with a cell population of 4.89 103 cells cm2 in a 5% CO2 humidified incubator at 37 °C. Confluent cell cultures grown in minimum essential medium (Euroclone) supplemented with 20% fetal bovine serum (Euroclone) and 2 non-essential amino acids (GIBCO), washed twice with phosphate-buffered saline (PBS; without Ca2+ and Mg2+) and then treated with 0.1% trypsin (Euroclone). Depleted cells were centrifuged at 141 rcf for 5 min. The resulting pellet was re-suspended in culture medium and then used for microcarrier seeding phase. The CultiSpher-G macroporous gelatine microcarrier (diameter 130–380 lm) (Percell Biolytica AB, Astorp, Sweden) is based on a highly crosslinked type A porcine-derived gelatine matrix. The mean internal pore size is 10–20 lm when rehydrated in PBS. All microcarriers used in this study were prepared according to the manufacturer’s instructions. Briefly, dry microcarriers were rehydrated in 1 PBS (without Ca2+ and Mg2+) for 1 h at room temperature. Without removing the PBS, the microcarriers were then autoclaved at 121 °C for 15 min and preincubated in cell culture medium at 4 °C overnight. Successively microcarriers were cell-seeded (105 cells per mg of microcarriers) in 250 ml of medium into a 500 ml siliconized spinner flask (Integra) using an intermittent stirring regime (30 min at 0 rpm, 5 min at 30 rpm) for 6 h. After seeding, the stirring speed was kept at a continuous 30 rpm for up to 4 days. All cultures were maintained at 37 °C in a humidified 5% CO2 incubator. 2.1.2. Cell adhesion assay After counting the free cells in the medium with a hemocytometer, the number of cells adhering to microbeads (cell/microbead ratio) was evaluated. Over the entire dynamic cell culture period in spinner flasks, 1 ml aliquots were collected each day for the cell adhesion assay on the microcarriers. Briefly, 200 ll of the same aliquots was transferred to a cell culture dish (w/2 mm grid Nunc) for microcarrier counting, after which the microcarrier suspension was placed in a new 1.5 ml tube. Before harvesting the attached cells by trypsinization, they were gently washed twice with PBS. The detached cells were then counted using a hemocytometer.
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2.1.3. Cell viability assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide) assay is a simple non-radioactive colorimetric assay used to measure cell cytotoxicity, proliferation or viability. MTT is cleaved by an enzyme during the respiration of mitochondria in living cells, generating formazan, which is a highly visible dark blue product. Each day 1 ml aliquots of the cell-attached microbead suspension were removed from the spinner flask, poured into bacterial dishes and incubated with 100 ll of MTT solution for 45 min at 37 °C for the viable cell staining.
2.1.4. Image analysis for tissue layer deposition evaluation To evaluate the cell and ECM deposition around the microcarriers during the spinner culture, the microcarriers diameter increase was monitored with Image J Software (National Institutes of Health, USA). Random samples were collected at 1 (group 1), 2 (group 2), 3 (group 3) and 4 (group 4) days of culture, and the images of 150 cell-seeded microbeads from a total of 20 visual fields were used for the analyses. Light microscopy images were transformed into binary images, and the microbeads’ surface and diameter size were evaluated. The aggregates of microbeads that eventually formed were not taken into account in the diameter distribution determination. The frequency (%) of diameters was counted and recorded in increments of 50 lm. As a control, the same analysis was carried out on microcarriers kept in cell-free culture at the 4 day time point. The expected value was evaluated for all diameter distributions.
2.1.5. Histological and immunohistochemical analyses At day 4 from the beginning of dynamic cell seeding, 1 ml of cell-seeded microbead suspension was fixed with 10% formaline for 24 h, dehydrated in an incremental series of alcohol (70%, 80%, 90% and 100%, each step 10 min) at room temperature and embedded in paraffin. Tissue sections, 5 lm thick, were stained with hematoxylin and eosin for morphological analysis and Masson’s trichrome for crosslinked collagen. The sections were mounted on coverslips and the morphological features of constructs were observed with a light microscope. For immunohistochemical analyses, deparaffinised 5 lm thick sections were stained with antibodies against type I collagen (Biodesign International, ME). The staining signal was developed with an avidin–peroxidase system (ABC kit; Vector Laboratories, Burlingame, CA).
2.1.6. Reverse transcription polymerase chain reaction (RT-PCR) analyses Expression of the type I collagen gene in 2-D cell samples and in lTPs collected after 4 days from spinner flasks was evaluated by RTPCR. mRNA was isolated (Tri Reagent, Sigma) and transcribed into cDNA and then amplified with a GeneAmp kit (GeneAmp Gold RNA PCR reagent kit; PE Biosystem). Based upon published sequences, specific primer sequences for bovine collagen a1 (I) were synthesized (Operon). The primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 50 -TGGAACTGATGAATGGGAGC-30 and 50 -GCAGCTTTTTCCTTTGTGGTG-30 (187 bp amplicon); those for type I collagen (a2) were 50 -TCCAAGGCAAAGAAGCAT-30 and 50 -GCAGCCATCTACAAGAAC-30 (297 bp amplicon). The PCR used 200 ng of cDNA, 0.15 lm of each primer, 0.8 mM dNTPs, 10 ll 5 PCR buffer, 1.75 mM MgCl2 and 0.5 U Taq polymerase in a total volume of 50 ll. The samples were denaturated first at 95 °C for 5 min, followed by 35 cycles of amplification of 95 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min. The PCR products were evaluated with cDNA and analyzed by electrophoresis on a 1% agarose gel; GAPDH primers (Operon) were used as a control with each cDNA.
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2.2. lTPs assembly
3. Results and discussion
To obtain a single and compact macrotissue of the desired shape and thickness, the lTP suspension was withdrawn from the spinner flask and transferred and cultured in an assembling chamber (Fig. 1B). The assembling chamber has a sandwich-like structure (Fig. 1Ba), in the middle of which is a silicon mould with four empty spaces (1 mm in thickness, 10 mm in diameter, Fig. 1Bb), where the lTPs assembly takes place. The silicon mould is delimited on both the top and bottom sides by two stainless steel rigid grids (Fig. 1Bc) characterized by a porous mesh (18 lm) that is able to retain the lTPs. Two polytetrafluoroethylene (PTFE) rings (Fig. 1Bd) are placed on the grids on both sides of the system and are fastened to each other by means of stainless steel screws, which close the system and ensure that the lTPs are retained. The system is autoclavable in each part. The lTP suspension was transferred from the spinner flask to a 50 ml Falcon centrifuge tube and, after settling, transferred by pipetting into the empty spaces of the silicon mould of the assembling chamber (Fig. 1B) to allow the assembly. Each opening was filled with approximately 13 mg of the lTPs. Furthermore, the assembling chamber was placed on the bottom of a spinner flask and completely surrounded by culture medium. The spinner was operated at 50 rpm and the medium was exchanged every 3 days. After 1 week of culture the assembling chamber was opened and the biohybrids were collected for histological analysis.
In this study, a bottom-up approach was proposed to build up 3-D tissue constructs in vitro by using lTPs as functional building units. The process philosophy is schematized in Fig. 1A. To realize the lTPs, a dynamic cell seeding on gelatine porous microcarriers was used. It has been suggested that the macroporous structure of CultiSpher microcarriers creates a suitable environment for the development of fibroblasts, as the cells within the interior will experience lower shear forces than those encountered with nonporous carriers [19–21]. Moreover, fibroblasts proliferating on macroporous gelatine based-microcarriers are able to synthesize a tissue with a dermal-like appearance [15]. The aim of this work was to exploit the neo-tissue layer generated on the lTPs as a biological bridge to build the final 3-D tissue construct. The spinner flask was loaded with 105 cell ml1 and 1 mg ml1 of microbeads in 250 ml of culture medium, corresponding to 100 cells per bead. The first 5 h of the seeding phase were characterized by intermittent stirring (5 min at 30 rpm, 30 min in static) to improve the cell-to-bead distribution and to obtain a lower proportion of unoccupied beads, as suggested in previous work [22]. The disappearance of free cells from the inoculated spinner cultures was considered to indicate the attachment of cells to the microcarriers. By determining the concentration of the fibroblasts in the culture medium during the intermittent seeding, it was possible to observe that the cell density in the culture medium (cells ml1) decreased by up to 60% after 6 h because of cell attachment to the microbeads and cell death. To investigate the viability of the cultured cells, MTT was used as a direct and simple assay. The analysis of cell viability via MTT revealed the progressive coating of microbeads with cells by
2.3. Statistical analysis Differences between groups were determined using a one-way analysis of variance with the Holm–Sidak test. Significance between groups was established for p < 0.05.
Fig. 1. Scheme of the bottom-up process (A); assembling chamber drawing (B), showing the configuration of the assembling chamber during the lTPs assembling process (a), the silicon mould with four empty spaces (b), the rigid mesh with 18 lm pore size (c) and the PTFE ring (d); lTPs loading (C).
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observing the increase in dark-stained spots present on the microbeads’ surface at different time points (data not shown). Moreover, a histological analysis was carried out to characterize the neo-tissue synthesized by fibroblasts adhered and proliferated on the gelatin microbeads surface. It was observed that some cells occupied the pores of the microbeads and a continuous cell and neo-matrix layer was present around the outer surface of the microbead at 48 h (Fig. 2A). The hybrid system composed of microbeads, cells and the neo-tissue layer is defined in this work as lTP. It was observed that lTPs were able to assemble due to the biological fusion between adjacent ECM layers, which constitute the ‘‘glue” that sticks the microparticles together (Fig. 2B). In order to characterize the biochemical nature of the neo-produced matrix layer, an immunohistochemical analysis of type I collagen expression was performed on the lTPs. Type I collagen is the main component of dermal tissue, and the performed analyses revealed the presence of this protein both in and around the lTPs (Fig. 2C). When the primary antibody for type I collagen was omitted, no staining was observed, confirming the specificity of the staining (data not shown). Furthermore, RT-PCR analysis showed that after 4 days of culture the type I collagen expression was higher in cells cultured in suspension conditions on gelatine microbeads than in 2-D monolayer culture (Fig. 2D). This behavior is probably due to the fact that a 3-D culture system, such as the microcarriers, can mimic the native environment that cells recognize as much more suitable for their activity. Taken together, these results highlight the effectiveness of dynamic cell seeding to maintain cell phenotype and promote ECM synthesis. The evolution of lTPs was monitored during spinner culture by both determining the cell/microbead ratio and measuring the thickness of the neo-tissue layer. The cell/microbead ratio
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was evaluated daily, and the results reported in Fig. 2E show that it increased from a value of 50 to a value of 170 after 4 days of culture due to the cell adhesion and proliferation on the microbeads’ surface. To monitor the extracellular matrix deposition on the microbeads, the cell-seeded microbeads’ diameter distribution was evaluated daily up to 4 days of spinner culture. As a control, the diameter distribution of naked microbeads at day 4 of culture was evaluated as well (Fig. 2F). As shown in Fig. 2F, the cell-seeded microbead distribution showed that the microbead frequency increased towards the higher diameter classes with culture time. Indeed, the expected value increased from 259 lm (group 1) to 409 lm (group 4), while the expected value of the control was 232 lm. The tissue layer deposition was thus observed to increase from 13 to 88 lm (Fig. 2F), indicating cell adhesion and proliferation and neo-matrix deposition on the gelatine microbeads. It is well known from the literature that microcarriers act as templates to propagate large numbers of cells. These can be retrieved for analytical purposes and cell transplantation, or as a cell delivery system for use in tissue engineering [23]. Additionally, under certain culture conditions, cells seeded on microcarriers have been shown to produce extracellular matrix that resembles many features of the tissue of origin, e.g. bovine chondrocytes [23,24], which act as delivery vehicles for the repair of bone and cartilage defects. In this work the surprising and very interesting results on the induced assembly lTPs after only 4 days of spinner culture suggest that this promising property can be exploited to realize and assemble 3-D biohybrids by placing the lTPs in mutual proximity in the assembling chamber (Fig. 3A). After 1 week of culture in the maturation chamber disc-shaped constructs (1 cm in diameter, 1 mm in thickness, Fig. 3B) of completely assembled lTPs
Fig. 2. Hematoxylin and eosin staining revealing the presence of a neo-ECM layer around a single lTP and cells inside its pores at 24 h of spinner culture (A) and among a plurality of lTPs allowing their aggregation after 96 h of spinner culture (B); immunohistochemical analysis performed on the lTPs revealing the presence of type I collagen in ECM (C); PCR analysis evidencing the presence of a higher type I collagen expression in cells cultured in spinner culture on gelatine microbeads than in 2-D monolayer culture; the 100 bp ladder (Life Technologies, Inc.) was used as a size standard (D); the cell/microbead ratio increased after 4 days of spinner culture. Values represent the mean and the standard deviation (n = 4; *p < 0.05) (E); diameter distribution of the cell-seeded microbeads and cell-free microbeads during culture time. Control represents naked microbeads diameter at day 4 of culture. Groups 1, 2, 3 and 4 represent cell-seeded microbeads diameter after 1, 2, 3 and 4 days of dynamic seeding, respectively (n = 20; group 4 vs. group 3, group 5 vs. group 1, group 5 vs. group 4, group 2 vs. group 3, group 4 vs. group 3 (p < 0,05)) (F). All bars are 200 lm.
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Fig. 3. Image of assembling chamber (A); macroscopic appearance of dermal-like construct after 1 week of culture (bar = 1 cm) (B); hematoxylin and eosin staining revealing the presence of a conspicuous neo-synthesized ECM inside the 3-D dermal-like construct (bar = 150 lm) (C and D); Masson trichromic staining showing collagen fiber in the neo-synthesized ECM (bar = 150 lm) (E); immunohistochemical analysis revealing synthesis of type I collagen (bar = 150 lm) (F).
were collected. The biohybrids obtained presented a homogeneous and compact aspect. Histological analysis assessed the presence of a viable neo-tissue with a conspicuous ECM among and inside gelatine microbeads (Fig. 3C and D). It is worth noting that the new synthesized tissue was characterized by a low cell/matrix ratio, a typical feature of connective tissues (e.g. dermis). Moreover, histologically, the biohybrid construct showed dermis-like tissue that consisted of an extracellular matrix around bovine fibroblasts, and revealed collagen fibers by Masson-trichrome staining (Fig. 3E). Finally, immunohistochemical staining demonstrated the presence of bovine type I collagen in the ECM of the biohybrid (Fig. 3F). Taken together, these results suggested that with our approach a 3-D dermal-like structure could be produced in which fibroblasts function like natural dermal fibroblasts by secreting collagen. The presence of the microbeads in the initial stage of the maturation process supplies mechanical support to the neo-tissue. Unlike with other bottom-up approaches [1,3,4], we obtained after just 1 week of maturation a 3-D biohybrid having the desired shape and thickness, that could be handled without any mechanical breakage. Furthermore, the lTPs proposed in this work can be injected into maturation chambers of the desired shape and subjected to dynamic culture conditions (i.e. perfusion) to produce thicker constructs, as needed [25]. However, a number of important considerations need to be raised. Some histological analyses of cell-laden microcarriers demonstrated that cells were able to colonize most pores and surfaces of microcarriers; however, some pores remained empty. This could indicate limited interconnectivity or that not all pores are accessible from the surface of the microcarriers [26,27]. This represents a possible limitation of the utility of CultiSpher-G microcarriers because the inability of cells to migrate into all pores reduces the total surface area available for cell expansion, matrix deposition and neo-tissue penetration. However, even though not all the pores of the macroporous microcarriers were taken up by newly formed tissue, we still believe that these carriers are more suitable than nonporous carriers because of the larger surface area of the beads for cell attachment and cell–material interaction. Histological analysis has also highlighted another possible limitation of the use of these commercial microcarriers: the commercial gelatine microbeads used herein are characterized by very low degradation rate. After
1 week in the assembling chamber, the beads maintained their wholeness, and their initial porous morphology was retained without any sign of degradation. Prolonged culture time has also been shown to lead to a contact inhibition phenomenon [28], which prevents the correct maturation and assembly of the tissue. To overcome these limitations, microbeads with engineered degradation rates could be used to allow faster replacement by the neo-matrix produced by cells and to obtain a macroconstruct completely made up of neo-synthesized tissues. Moreover, bovine fibroblasts were used herein, but different anchorage-dependent cell types could be used to allow the realization of different kind of lTPs. In this fashion, multicellular biohybrid constructs could be produced that would generate tissue-like structures for a wide range of applications. 4. Conclusion We have demonstrated in this work that functional building blocks for bio-fabrication of complex, 3-D tissue can be realized by using cell-seeded porous microbeads. The results show that under appropriate cell culture conditions it is possible to induce the assembly of lTPs into single compact tissue constructs, which in this specific case can be defined as a dermal-like equivalent. Finally, as tissue printing [2,29] is becoming a powerful bottom-up approach to engineering complex 3-D tissue, the lTPs proposed herein could be used as building blocks in this technology. Acknowledgments The authors are grateful for financial support provided by the EU community, DERMAGENESIS U.E. COLL-CT2003-500224 and by Italian Public Instruction Ministry, Tissuenet n. RBPRO5RSM2. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2 and 3, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2010.01.026.
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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat
Brief communication
Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors Carmela Palmiero a,c, Giorgia Imparato a,c, Francesco Urciuolo b,c, Paolo Netti a,c,* a
Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, P. Le Tecchio 80, 80125 Naples, Italy Institute of Composite and Biomedical Materials (IMCB), National Council Research (CNR), P. Le Tecchio 80, 80125 Naples, Italy c Italian Institute of Technology (IIT), Via Morego 30, 16163 Genoa, Italy b
a r t i c l e
i n f o
Article history: Received 9 October 2009 Received in revised form 29 December 2009 Accepted 19 January 2010 Available online 25 January 2010 Keywords: Tissue engineering Microcarriers Bio-fabrication Dermis
a b s t r a c t Tissue-engineered constructs can be fabricated by the assembly of smaller building blocks in order to mimic much of the native biology that is often made from repeating functional units. Our aim was to realize a three-dimensional (3-D) tissue-like construct in vitro by inducing the assembly of functional micrometric tissue precursors (lTPs). lTPs were obtained by dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. During the dynamic seeding, cells adhered, proliferated and synthesized a thin layer of extracellular matrix (ECM) in and around the macroporous beads, generating the lTPs. The analysis showed that the ECM produced was rich in type I collagen. The cells and ECM layer around the lTPs allowed their biological sintering via cell–cell and cell–matrix interaction after only a few days of dynamic seeding. The assembling ability of lTPs was exploited by placing them in a maturation chamber. After 1 week of culture disc-shaped constructs (1 cm in diameter, 1 mm in thickness) of completely assembled lTPs were collected. The biohybrid obtained presented both a homogeneous and compact aspect. Moreover, histological and immunohistochemical analyses revealed an abundant ECM, rich in type I collagen, interconnecting the lTPs. The results obtained in this survey pave the way to realizing a 3-D dermal tissue equivalent by means of a bottom-up tissue engineering approach. Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction Most living tissues are composed of repeating units on the scale of hundreds of microns, which are ensembles of different cell types with well defined three-dimensional (3-D) micro-architectures and tissue-specific, functional properties. To generate engineered tissues, the recreation of these structural features is of great importance in enabling the resulting function [1]. However, the classical tissue engineering approach, based on seeding cells into a preformed, porous and biodegradable scaffold to recreate the natural tissue complex structure, faces serious hurdles. Selection of the ideal biomaterial scaffold for a given cell type is problematic and has been accomplished to date mostly by trial and error. Even if the right biomaterial is available, achieving a high enough cell density and the homogeneous cell distribution necessary to construct a viable tissue is extremely time consuming. Furthermore, preshaping the scaffold may present further difficulties [2]. To overcome these limitations, recent efforts [3–11] have been concentrated on scaffold-less tissue engineering and bot-
* Corresponding author. Address: Interdisciplinary Research Centre on Biomaterials (CRIB), University of Naples Federico II, P. Le Tecchio 80, 80125 Naples, Italy. Tel.: +39 081 7682408; fax: +39 081 7682404. E-mail address: [email protected] (P. Netti).
tom-up approaches aimed at generating a larger tissue construct by the assembly of smaller building blocks, which mimics the in vivo tissue structure of repeating functional units. A similar strategy was proposed by Du et al. [1], who used a bottom-up approach to direct the assembly of cell-laden microgels to generate 3D tissue with tunable microarchitecture and complexity. McGuigan and Sefton [3] proposed another interesting use of modular components for generating tissue. In their approach, rod-shaped collagen microgels that were seeded with HepG2 hepatocytes on the inside and endothelial cells on the surface were ‘‘packed” together within a bioreactor and perfused with medium. Further examples of constructs resulting from a bottom-up approach include beating cardiac sheets, generated by the stacking of sheets of cells for patches obtained by means of ‘‘cell sheet technology” [4]. In this technology, a cell sheet produced by a wellestablished 2-D cell culture system plays the role of a building block of organ-like structures. By using thermoresponsive culture dishes, cell sheets are easily harvested as intact monolayers along with their deposited extracellular matrix (ECM) and can be layered on top of one another to create a 3-D construct, such as thick cardiac muscle [12–15]. The use of cell sheets has the advantage that an entirely natural neo-tissue assembled from cells with a mature ECM can be engineered. However, this technology has a number of shortcomings,
1742-7061/$ - see front matter Ó 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2010.01.026
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such as handling difficulties with the cell sheets and the limited number of cell sheets that can be effectively layered without core ischemia or hypoxia [4,16,17]. In this paper, we propose a tissue engineering strategy that allows the production of viable thick 3-D tissues with a compact ECM by using a bottom-up approach. Micrometric tissue precursors (lTPs) were obtained by means of dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. Numerous studies have illustrated that the particle cultivation technique is more effective than cell culture on flat substrates such as culture dishes [18,19]. In this study, we observed that under optimal culture conditions cells were able to adhere, proliferate and in particular synthesize ECM components to form a thin layer of tissue around the microbeads, generating microscale tissue units which we refer to as lTPs. The lTPs have been used as an ideal ‘‘material” for bio-fabrication of 3-D tissue constructs. Indeed, we demonstrate that lTPs can be assembled, in an appropriate assembling chamber, by means of the tissue layers surrounding them, which allow their fusion through cell–cell and cell–matrix interactions. Following this strategy, a 3-D functional dermal tissue equivalent has been realized. 2. Materials and methods 2.1. Sampling and characterization of lTPs 2.1.1. Cell culture and dynamic cell seeding Primary bovine dermal fibroblasts (BF-AG10385 Coriell) were propagated in monolayer culture in a 150 mm Petri dish with a cell population of 4.89 103 cells cm2 in a 5% CO2 humidified incubator at 37 °C. Confluent cell cultures grown in minimum essential medium (Euroclone) supplemented with 20% fetal bovine serum (Euroclone) and 2 non-essential amino acids (GIBCO), washed twice with phosphate-buffered saline (PBS; without Ca2+ and Mg2+) and then treated with 0.1% trypsin (Euroclone). Depleted cells were centrifuged at 141 rcf for 5 min. The resulting pellet was re-suspended in culture medium and then used for microcarrier seeding phase. The CultiSpher-G macroporous gelatine microcarrier (diameter 130–380 lm) (Percell Biolytica AB, Astorp, Sweden) is based on a highly crosslinked type A porcine-derived gelatine matrix. The mean internal pore size is 10–20 lm when rehydrated in PBS. All microcarriers used in this study were prepared according to the manufacturer’s instructions. Briefly, dry microcarriers were rehydrated in 1 PBS (without Ca2+ and Mg2+) for 1 h at room temperature. Without removing the PBS, the microcarriers were then autoclaved at 121 °C for 15 min and preincubated in cell culture medium at 4 °C overnight. Successively microcarriers were cell-seeded (105 cells per mg of microcarriers) in 250 ml of medium into a 500 ml siliconized spinner flask (Integra) using an intermittent stirring regime (30 min at 0 rpm, 5 min at 30 rpm) for 6 h. After seeding, the stirring speed was kept at a continuous 30 rpm for up to 4 days. All cultures were maintained at 37 °C in a humidified 5% CO2 incubator. 2.1.2. Cell adhesion assay After counting the free cells in the medium with a hemocytometer, the number of cells adhering to microbeads (cell/microbead ratio) was evaluated. Over the entire dynamic cell culture period in spinner flasks, 1 ml aliquots were collected each day for the cell adhesion assay on the microcarriers. Briefly, 200 ll of the same aliquots was transferred to a cell culture dish (w/2 mm grid Nunc) for microcarrier counting, after which the microcarrier suspension was placed in a new 1.5 ml tube. Before harvesting the attached cells by trypsinization, they were gently washed twice with PBS. The detached cells were then counted using a hemocytometer.
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2.1.3. Cell viability assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide) assay is a simple non-radioactive colorimetric assay used to measure cell cytotoxicity, proliferation or viability. MTT is cleaved by an enzyme during the respiration of mitochondria in living cells, generating formazan, which is a highly visible dark blue product. Each day 1 ml aliquots of the cell-attached microbead suspension were removed from the spinner flask, poured into bacterial dishes and incubated with 100 ll of MTT solution for 45 min at 37 °C for the viable cell staining.
2.1.4. Image analysis for tissue layer deposition evaluation To evaluate the cell and ECM deposition around the microcarriers during the spinner culture, the microcarriers diameter increase was monitored with Image J Software (National Institutes of Health, USA). Random samples were collected at 1 (group 1), 2 (group 2), 3 (group 3) and 4 (group 4) days of culture, and the images of 150 cell-seeded microbeads from a total of 20 visual fields were used for the analyses. Light microscopy images were transformed into binary images, and the microbeads’ surface and diameter size were evaluated. The aggregates of microbeads that eventually formed were not taken into account in the diameter distribution determination. The frequency (%) of diameters was counted and recorded in increments of 50 lm. As a control, the same analysis was carried out on microcarriers kept in cell-free culture at the 4 day time point. The expected value was evaluated for all diameter distributions.
2.1.5. Histological and immunohistochemical analyses At day 4 from the beginning of dynamic cell seeding, 1 ml of cell-seeded microbead suspension was fixed with 10% formaline for 24 h, dehydrated in an incremental series of alcohol (70%, 80%, 90% and 100%, each step 10 min) at room temperature and embedded in paraffin. Tissue sections, 5 lm thick, were stained with hematoxylin and eosin for morphological analysis and Masson’s trichrome for crosslinked collagen. The sections were mounted on coverslips and the morphological features of constructs were observed with a light microscope. For immunohistochemical analyses, deparaffinised 5 lm thick sections were stained with antibodies against type I collagen (Biodesign International, ME). The staining signal was developed with an avidin–peroxidase system (ABC kit; Vector Laboratories, Burlingame, CA).
2.1.6. Reverse transcription polymerase chain reaction (RT-PCR) analyses Expression of the type I collagen gene in 2-D cell samples and in lTPs collected after 4 days from spinner flasks was evaluated by RTPCR. mRNA was isolated (Tri Reagent, Sigma) and transcribed into cDNA and then amplified with a GeneAmp kit (GeneAmp Gold RNA PCR reagent kit; PE Biosystem). Based upon published sequences, specific primer sequences for bovine collagen a1 (I) were synthesized (Operon). The primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were 50 -TGGAACTGATGAATGGGAGC-30 and 50 -GCAGCTTTTTCCTTTGTGGTG-30 (187 bp amplicon); those for type I collagen (a2) were 50 -TCCAAGGCAAAGAAGCAT-30 and 50 -GCAGCCATCTACAAGAAC-30 (297 bp amplicon). The PCR used 200 ng of cDNA, 0.15 lm of each primer, 0.8 mM dNTPs, 10 ll 5 PCR buffer, 1.75 mM MgCl2 and 0.5 U Taq polymerase in a total volume of 50 ll. The samples were denaturated first at 95 °C for 5 min, followed by 35 cycles of amplification of 95 °C for 1 min, 57 °C for 1 min and 72 °C for 1 min. The PCR products were evaluated with cDNA and analyzed by electrophoresis on a 1% agarose gel; GAPDH primers (Operon) were used as a control with each cDNA.
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2.2. lTPs assembly
3. Results and discussion
To obtain a single and compact macrotissue of the desired shape and thickness, the lTP suspension was withdrawn from the spinner flask and transferred and cultured in an assembling chamber (Fig. 1B). The assembling chamber has a sandwich-like structure (Fig. 1Ba), in the middle of which is a silicon mould with four empty spaces (1 mm in thickness, 10 mm in diameter, Fig. 1Bb), where the lTPs assembly takes place. The silicon mould is delimited on both the top and bottom sides by two stainless steel rigid grids (Fig. 1Bc) characterized by a porous mesh (18 lm) that is able to retain the lTPs. Two polytetrafluoroethylene (PTFE) rings (Fig. 1Bd) are placed on the grids on both sides of the system and are fastened to each other by means of stainless steel screws, which close the system and ensure that the lTPs are retained. The system is autoclavable in each part. The lTP suspension was transferred from the spinner flask to a 50 ml Falcon centrifuge tube and, after settling, transferred by pipetting into the empty spaces of the silicon mould of the assembling chamber (Fig. 1B) to allow the assembly. Each opening was filled with approximately 13 mg of the lTPs. Furthermore, the assembling chamber was placed on the bottom of a spinner flask and completely surrounded by culture medium. The spinner was operated at 50 rpm and the medium was exchanged every 3 days. After 1 week of culture the assembling chamber was opened and the biohybrids were collected for histological analysis.
In this study, a bottom-up approach was proposed to build up 3-D tissue constructs in vitro by using lTPs as functional building units. The process philosophy is schematized in Fig. 1A. To realize the lTPs, a dynamic cell seeding on gelatine porous microcarriers was used. It has been suggested that the macroporous structure of CultiSpher microcarriers creates a suitable environment for the development of fibroblasts, as the cells within the interior will experience lower shear forces than those encountered with nonporous carriers [19–21]. Moreover, fibroblasts proliferating on macroporous gelatine based-microcarriers are able to synthesize a tissue with a dermal-like appearance [15]. The aim of this work was to exploit the neo-tissue layer generated on the lTPs as a biological bridge to build the final 3-D tissue construct. The spinner flask was loaded with 105 cell ml1 and 1 mg ml1 of microbeads in 250 ml of culture medium, corresponding to 100 cells per bead. The first 5 h of the seeding phase were characterized by intermittent stirring (5 min at 30 rpm, 30 min in static) to improve the cell-to-bead distribution and to obtain a lower proportion of unoccupied beads, as suggested in previous work [22]. The disappearance of free cells from the inoculated spinner cultures was considered to indicate the attachment of cells to the microcarriers. By determining the concentration of the fibroblasts in the culture medium during the intermittent seeding, it was possible to observe that the cell density in the culture medium (cells ml1) decreased by up to 60% after 6 h because of cell attachment to the microbeads and cell death. To investigate the viability of the cultured cells, MTT was used as a direct and simple assay. The analysis of cell viability via MTT revealed the progressive coating of microbeads with cells by
2.3. Statistical analysis Differences between groups were determined using a one-way analysis of variance with the Holm–Sidak test. Significance between groups was established for p < 0.05.
Fig. 1. Scheme of the bottom-up process (A); assembling chamber drawing (B), showing the configuration of the assembling chamber during the lTPs assembling process (a), the silicon mould with four empty spaces (b), the rigid mesh with 18 lm pore size (c) and the PTFE ring (d); lTPs loading (C).
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observing the increase in dark-stained spots present on the microbeads’ surface at different time points (data not shown). Moreover, a histological analysis was carried out to characterize the neo-tissue synthesized by fibroblasts adhered and proliferated on the gelatin microbeads surface. It was observed that some cells occupied the pores of the microbeads and a continuous cell and neo-matrix layer was present around the outer surface of the microbead at 48 h (Fig. 2A). The hybrid system composed of microbeads, cells and the neo-tissue layer is defined in this work as lTP. It was observed that lTPs were able to assemble due to the biological fusion between adjacent ECM layers, which constitute the ‘‘glue” that sticks the microparticles together (Fig. 2B). In order to characterize the biochemical nature of the neo-produced matrix layer, an immunohistochemical analysis of type I collagen expression was performed on the lTPs. Type I collagen is the main component of dermal tissue, and the performed analyses revealed the presence of this protein both in and around the lTPs (Fig. 2C). When the primary antibody for type I collagen was omitted, no staining was observed, confirming the specificity of the staining (data not shown). Furthermore, RT-PCR analysis showed that after 4 days of culture the type I collagen expression was higher in cells cultured in suspension conditions on gelatine microbeads than in 2-D monolayer culture (Fig. 2D). This behavior is probably due to the fact that a 3-D culture system, such as the microcarriers, can mimic the native environment that cells recognize as much more suitable for their activity. Taken together, these results highlight the effectiveness of dynamic cell seeding to maintain cell phenotype and promote ECM synthesis. The evolution of lTPs was monitored during spinner culture by both determining the cell/microbead ratio and measuring the thickness of the neo-tissue layer. The cell/microbead ratio
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was evaluated daily, and the results reported in Fig. 2E show that it increased from a value of 50 to a value of 170 after 4 days of culture due to the cell adhesion and proliferation on the microbeads’ surface. To monitor the extracellular matrix deposition on the microbeads, the cell-seeded microbeads’ diameter distribution was evaluated daily up to 4 days of spinner culture. As a control, the diameter distribution of naked microbeads at day 4 of culture was evaluated as well (Fig. 2F). As shown in Fig. 2F, the cell-seeded microbead distribution showed that the microbead frequency increased towards the higher diameter classes with culture time. Indeed, the expected value increased from 259 lm (group 1) to 409 lm (group 4), while the expected value of the control was 232 lm. The tissue layer deposition was thus observed to increase from 13 to 88 lm (Fig. 2F), indicating cell adhesion and proliferation and neo-matrix deposition on the gelatine microbeads. It is well known from the literature that microcarriers act as templates to propagate large numbers of cells. These can be retrieved for analytical purposes and cell transplantation, or as a cell delivery system for use in tissue engineering [23]. Additionally, under certain culture conditions, cells seeded on microcarriers have been shown to produce extracellular matrix that resembles many features of the tissue of origin, e.g. bovine chondrocytes [23,24], which act as delivery vehicles for the repair of bone and cartilage defects. In this work the surprising and very interesting results on the induced assembly lTPs after only 4 days of spinner culture suggest that this promising property can be exploited to realize and assemble 3-D biohybrids by placing the lTPs in mutual proximity in the assembling chamber (Fig. 3A). After 1 week of culture in the maturation chamber disc-shaped constructs (1 cm in diameter, 1 mm in thickness, Fig. 3B) of completely assembled lTPs
Fig. 2. Hematoxylin and eosin staining revealing the presence of a neo-ECM layer around a single lTP and cells inside its pores at 24 h of spinner culture (A) and among a plurality of lTPs allowing their aggregation after 96 h of spinner culture (B); immunohistochemical analysis performed on the lTPs revealing the presence of type I collagen in ECM (C); PCR analysis evidencing the presence of a higher type I collagen expression in cells cultured in spinner culture on gelatine microbeads than in 2-D monolayer culture; the 100 bp ladder (Life Technologies, Inc.) was used as a size standard (D); the cell/microbead ratio increased after 4 days of spinner culture. Values represent the mean and the standard deviation (n = 4; *p < 0.05) (E); diameter distribution of the cell-seeded microbeads and cell-free microbeads during culture time. Control represents naked microbeads diameter at day 4 of culture. Groups 1, 2, 3 and 4 represent cell-seeded microbeads diameter after 1, 2, 3 and 4 days of dynamic seeding, respectively (n = 20; group 4 vs. group 3, group 5 vs. group 1, group 5 vs. group 4, group 2 vs. group 3, group 4 vs. group 3 (p < 0,05)) (F). All bars are 200 lm.
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Fig. 3. Image of assembling chamber (A); macroscopic appearance of dermal-like construct after 1 week of culture (bar = 1 cm) (B); hematoxylin and eosin staining revealing the presence of a conspicuous neo-synthesized ECM inside the 3-D dermal-like construct (bar = 150 lm) (C and D); Masson trichromic staining showing collagen fiber in the neo-synthesized ECM (bar = 150 lm) (E); immunohistochemical analysis revealing synthesis of type I collagen (bar = 150 lm) (F).
were collected. The biohybrids obtained presented a homogeneous and compact aspect. Histological analysis assessed the presence of a viable neo-tissue with a conspicuous ECM among and inside gelatine microbeads (Fig. 3C and D). It is worth noting that the new synthesized tissue was characterized by a low cell/matrix ratio, a typical feature of connective tissues (e.g. dermis). Moreover, histologically, the biohybrid construct showed dermis-like tissue that consisted of an extracellular matrix around bovine fibroblasts, and revealed collagen fibers by Masson-trichrome staining (Fig. 3E). Finally, immunohistochemical staining demonstrated the presence of bovine type I collagen in the ECM of the biohybrid (Fig. 3F). Taken together, these results suggested that with our approach a 3-D dermal-like structure could be produced in which fibroblasts function like natural dermal fibroblasts by secreting collagen. The presence of the microbeads in the initial stage of the maturation process supplies mechanical support to the neo-tissue. Unlike with other bottom-up approaches [1,3,4], we obtained after just 1 week of maturation a 3-D biohybrid having the desired shape and thickness, that could be handled without any mechanical breakage. Furthermore, the lTPs proposed in this work can be injected into maturation chambers of the desired shape and subjected to dynamic culture conditions (i.e. perfusion) to produce thicker constructs, as needed [25]. However, a number of important considerations need to be raised. Some histological analyses of cell-laden microcarriers demonstrated that cells were able to colonize most pores and surfaces of microcarriers; however, some pores remained empty. This could indicate limited interconnectivity or that not all pores are accessible from the surface of the microcarriers [26,27]. This represents a possible limitation of the utility of CultiSpher-G microcarriers because the inability of cells to migrate into all pores reduces the total surface area available for cell expansion, matrix deposition and neo-tissue penetration. However, even though not all the pores of the macroporous microcarriers were taken up by newly formed tissue, we still believe that these carriers are more suitable than nonporous carriers because of the larger surface area of the beads for cell attachment and cell–material interaction. Histological analysis has also highlighted another possible limitation of the use of these commercial microcarriers: the commercial gelatine microbeads used herein are characterized by very low degradation rate. After
1 week in the assembling chamber, the beads maintained their wholeness, and their initial porous morphology was retained without any sign of degradation. Prolonged culture time has also been shown to lead to a contact inhibition phenomenon [28], which prevents the correct maturation and assembly of the tissue. To overcome these limitations, microbeads with engineered degradation rates could be used to allow faster replacement by the neo-matrix produced by cells and to obtain a macroconstruct completely made up of neo-synthesized tissues. Moreover, bovine fibroblasts were used herein, but different anchorage-dependent cell types could be used to allow the realization of different kind of lTPs. In this fashion, multicellular biohybrid constructs could be produced that would generate tissue-like structures for a wide range of applications. 4. Conclusion We have demonstrated in this work that functional building blocks for bio-fabrication of complex, 3-D tissue can be realized by using cell-seeded porous microbeads. The results show that under appropriate cell culture conditions it is possible to induce the assembly of lTPs into single compact tissue constructs, which in this specific case can be defined as a dermal-like equivalent. Finally, as tissue printing [2,29] is becoming a powerful bottom-up approach to engineering complex 3-D tissue, the lTPs proposed herein could be used as building blocks in this technology. Acknowledgments The authors are grateful for financial support provided by the EU community, DERMAGENESIS U.E. COLL-CT2003-500224 and by Italian Public Instruction Ministry, Tissuenet n. RBPRO5RSM2. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figures 1, 2 and 3, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi:10.1016/j.actbio. 2010.01.026.
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