We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.
Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.
One general problem in tissue engineering applications is to yield a high cell mass with the correct differentiation phenotype at the location of need. The application of microcarriers to address this issue started in 1967 with increasing significance to date in fields such as orthopaedic tissue engineering for large-scale generation of skin, bone, cartilage, and tendons1. They allow the handling of adherent cultures in ways similar to that of suspension cultures2 by expanding cells on microscale three-dimensional (3D) substrates. Thereby cells experience a homogeneous nutrient supply and cell-matrix interactions that lead to better maintenance of in vivo3,4 differentiation which is often lost over time in 2D approaches5. A higher surface-to-volume ratio – eventually leading to higher cell yields6,7, higher gas and nutrient exchange rates comparing to static systems8, the possibility to regulate and subject the culture to physical stimuli9, and the potential for scaling up of the expansion process7 are further advantages. Several features such as diameter, density, porosity, surface charge, and adhesion properties10,11 distinguish the different commercially available micro- and macro-carriers. However, one of the main advantage is their delivery potential as microtissues to site defect or demand.
For applications of the microcarrier technology in bone tissue engineering, we illustrated in a previous report12 the production of a new microcarrier type constituted of a recombinant collagen I peptide (RCP, commercially available as Cellnest). This new microcarrier allows the GMP-compliant up scaling of scaffold and cell production, as needed for cell delivery in a clinical scenario. In this context, tuning of scaffold stability, degradation rate, and surface properties through proper choice of a suited crosslinking strategy allows to adapt the technique to the selected application, cell type of interest or target tissue13. In particular, the potential employment of this microcarrier as an injectable cell delivery system for therapeutic application14 makes them particularly interesting in a clinical setting.
In this paper, we therefore illustrate the culturing procedure for the isolation and expansion of human bone marrow-derived mesenchymal stromal cells (hBMSCs) and human dermal microvascular endothelial cells (HDMECs) on collagen-I-based recombinant peptide-based microcarriers, and their preparation for delivery in a clinical setting. Furthermore, we describe additional protocols useful for the maintenance of cell viability upon implantation.
Cell viability after implantation is in fact strongly dependent on vascularization15,16,17, which ensures exchange of oxygen and nutrients and facilitates waste removal. Bioreactors constitute one approach to overcome vascularization challenges in tissue engineering and maintain cell viability, through perfusion of culture medium providing thereby oxygen and nutrients18. Here, we illustrate an in vitro method to evaluate the migration capability of microvascular endothelial cells from the RCP microcarriers to a biomatrix and their ability to contribute to the de novo vascularization and angiogenesis. This biomatrix is a decellularized segment of porcine jejunum termed BioVaSc (Biological Vascularized Scaffold), rich in collagen and elastin and with preserved vascular structures, which includes a feeding artery and a draining vein19 that has been applied for implantation issues20.
hBMSCs were isolated from the femur head of osteoarthritis patients undergoing femur head replacement surgery. The procedure was performed under the approval of the Local Ethics Committee of the University of Wuerzburg and informed consent of the patients. Primary microvascular endothelial cells were isolated from foreskin biopsies of juvenile donors. Their legal representative(s) provided full informed consent in writing. The study was approved by the local ethical board of the University of Wuerzburg (vote 182/10).
1. Isolation of hBMSCs and HDMECs
2. Set-up and sterilization of spinner flasks, RCP microcarriers, and bioreactor
3. Culture of hBMSCs and HDMECs on RCP macroporous microcarriers
NOTE: The HDMECs used to seed the RCP microcarriers were labeled with RFP by lentiviral transduction. This protocol was modified after a previously published protocol5.
4. DNA content, SEM analysis, live dead staining, and sprouting assay
5. Biomatrix seeding and start of bioreactor system for HDMECs
6. Addition of RFP-HDMECs to the biomatrix
7. Analysis and read-outs of bioreactor cultures
As shown in Figure 1A, we obtained high number of viable cells on the RCP microcarriers after 7 days of culture, determined by live/dead staining. Those results were confirmed by SEM analysis, in which completely colonized microcarriers were observed around the pores, partly overgrowing them (Figure 1B). On the other hand, experiments in which cells were not evenly seeded resulted in several empty microcarriers. Failed experiments are characterized by an abnormally high number of dead cells that should not be over 10% of the total cell amount (Figure 1C). Moreover, DNA content quantification of HDMECs showed an increase of dsDNA over time (Figure 1D).
Additionally, RFP-HDMECs were able to maintain their functionality after culture on RCP microcarriers, as shown in Figure 2, where the cells adopted a sprouting phenotype when cultured in a collagen gel.
Furthermore, we used RFP-HDMECs-colonized RCP microcarriers to re-seed the lumen of the biomatrix BioVaSc. For this, we employed a bioreactor system for physiological perfusion of vascularized tissue equivalents22 (Figure 3C) in which we cut-open the biomatrix and placed it in a polycarbonate frame, so that the lumen was exposed in an even set-up (Figure 3B).
After 21 days of bioreactor cultures, metabolically active cells were present both on the preserved vasculature of the biomatrix as well as on the RCP microcarriers (Figure 4A and Figure 3B). Failed experiments or a wrong choice of slices during sectioning of the histological samples can lead to absence of colonizing endothelial cells in the lumen of the vessels of the biomatrix or on the microcarriers. These results could be confirmed by SEM analysis of the biomatrix sections, where it could be observed that some areas of the RCP microcarriers were still covered by cells and that the RCP microcarriers were in close proximity to the biomatrix (Figure 4C and 4D).
Finally, H&E staining confirmed the presence of HDMECs on the biomatrix, specifically in the vascular structures (Figure 5A). When observed under fluorescence microscope, the cells colonizing the vascular structures of the biomatrix resulted to be RFP-HDMECs (Figure 5B), indicating the migration of the cells from the RCP microcarriers to the biomatrix. Some RFP-HDMECs were also observed on the RCP microcarriers (Figure 5C and 5D). In experiments in which cells did not survived due to altered culturing conditions (e.g. shorter culture times), they could not be detected inside the vascular structure with none of the previously reported techniques (Figure 5E and 5F).
Figure 1: Culture of HDMECs and hBMSCs on RCP microcarriers. (A, C) live/dead staining after 7 days of dynamic cultures. (B) SEM analysis of HDMECs cultured on RCP microcarriers after 7 days of dynamic cultures. (D) DNA content determined by PicoGreen assay kit. Data expressed as mean ± standard deviation (n=3). Scale bars: 200 µm for A, 88 µm for B, and 100 µm for C. Please click here to view a larger version of this figure.
Figure 2: Sprouting assay. Sprouts of RFP-HDMECs can be observed under bright-field (A) and fluorescence microscopy (B), emerging from the colonized RCP microcarrier after 24 h of culture in a collagen gel. (C) Overlay image. Scale bars: 100 µm. Please click here to view a larger version of this figure.
Figure 3: Biomatrix and bioreactor set-up. (A) Preparation of the biomatrix and mounting on the polycarbonate frame (B). (C) Bioreactor set-up. The vessel is connected to the medium reservoir and the pressure bottle through silicon tubes, and the whole system is connected to a peristaltic pump. The pressure is measured through a pressure sensor. AI: arterial inlet; VO: venous outlet. This figure has been modified from Groeber et al.22 Please click here to view a larger version of this figure.
Figure 4: Bioreactor read-out. MTT assay performed on a biomatrix section after 21 days of culture, in which metabolically active cells were observed in the preserved vasculature of the biomatrix (A) as well as on the RCP microcarriers (B). (C, D) SEM images of RCP microcarriers on a biomatrix section. Scale bars: 0.28 cm for A, 0.45 cm for B, 47 µm for C, and 225 µm for D. Please click here to view a larger version of this figure.
Figure 5: H&E staining & fluorescence images. (A, C, E) H&E staining. (B, D, F) After 21 days of culture, RFP-HDMECs were observed inside of the vasculature of the biomatrix (arrows) as well as on the RCP microcarriers (arrow heads). Scale bars: 50 µm for A-D and 30 µm for E and F. Please click here to view a larger version of this figure.
One main goal of microcarrier is the expansion of cells while maintaining their differentiation in order to deliver cells to the place of need. The represented method introduce RCP microcarriers where cells were able to attach, proliferate, and colonize the microcarriers with high cell density. This was observed by live/dead staining, in which more than 90% of viable cells were detected while only few dead cells were obtained after 7 days of dynamic cultures. Likewise, the SEM images confirmed that the cells covered the entire surface of the microcarriers after 7 days of cultures.
To ensure cell survival in 3D models, it is important to maintain the supply of oxygen and nutrients to the cells and allow removal of waste substances. Blood vessels are the responsible structures of this exchange process, in which endothelial cells play a key role since they are the cells lining the inner part of blood vessels23. They have the capability to proliferate, migrate, adhere, sprout, and form vessel-like structures24. These properties were maintained after culture of RFP-HDMECs on the RCP microcarriers, since it was observed that the cells were able to adopt the sprouting phenotype (see Figure 2). We could prove here that these colonized RCP microcarriers were effectively delivering primary endothelial cells to re-seed the lumen of former vessels of the BioVaSc biomatrix. This suggests our system to be suitable to improve the vascularization of tissue engineered implants for clinical applications. Derivatives of this matrix have been successfully used in vascularization approaches25, as well as in in vitro tumor test sytems21,26,27,28 and for the production of skin equivalents as alternatives to animal experimentation29. Here it is used in an open, flattened set-up and placed in a perfusion bioreactor as applied for the generation of tissue equivalents22.
Bioreactors have been used in tissue engineering to produce tissue equivalents that could help ease the demand of organs while reducing the associated risks like rejection and morbidity30. However, producing tissue-like constructs is a very complex process that involves the use of tissue-specific cell types, a suitable scaffold and appropriate growth factors that allow the proper differentiation and assembling into 3D tissues. One major drawback of the engineered constructs is the lack of a proper vascular network that supports cells survival both in vitro and in vivo17. Here we combine the RCP-colonized microcarriers with a decellularized matrix in a bioreactor model, providing the cell type required, a scaffold that allows cell adhesion and vessel formation and a specific culture medium that provides the essential factors for the cells to proliferate and maintain their functional properties. A significant result of this model is the ability of the HDMECs to migrate from the RCP microcarriers to the scaffold without any additional detachment or digestion as necessary in other approaches31. Afterwards, they colonized specifically the vascular structures of the matrix, proving the concept that macroporous microcarriers can be used as cell delivery system for Regenerative Medicine purposes.
Altogether, this protocol represents a promising strategy in regenerative medicine for obtaining a maximized cell expansion and it could serve as cell delivery to implantation sites, where it could improve vascularization support for tissue engineered grafts.
The authors have nothing to disclose.
The research leading to these results has received funding from the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement n° 607051 (BIO-INSPIRE). We thank Carolien van Spreuwel-Goossens from Fujifilm Manufacturing Europe B.V., for the technical assistance during RCP manufacturing, and Werner Stracke from Fraunhofer Institute for Silicate Research ISC, for assistance with the SEM analysis.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) | Serva Electrophoresis GmbH | 20395.01 | |
4’,6-Diamidino-2-phenylindoldihydrochloride (DAPI) | Sigma-Aldrich | D9542 | |
Acetic acid 100% | Sigma-Aldrich | 533,001 | |
Analytical balance Kern EG 2200-2NM | Kern & Sohn GmbH | ||
Ascorbate-2-phosphate | Sigma-Aldrich | A8960 | |
Bioreactor | Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany | ||
Bright field microscope Axiovert 40C | Carl Zeiss AG | ||
Cellnest | Fujifilm | ||
Centrifuge tubes (15 mL, 50 mL) | Greiner Bio-One | ||
Collagen R solution 0,4% | Serva Electrophoresis GmbH | 47254.01 | |
DMEM-F12 | Gibco | 11320-033 | |
Dulbecco's Phosphate Buffered Saline | Sigma-Aldrich | D8537 | Modified, without calcium chloride and magnesium chloride |
Eosin 1% | Morphisto | 10177.01000 | |
Ethanol 96% | Carl Roth GmbH | T171.4 | Denatured |
Fetal calf serum (FCS) | Bio&SELL | FCS.ADD.0500 | not heat-inactivated |
Fluorescence microscope BZ-9000 | Keyence | ||
Haematoxylin | Morphisto | 10231.01000 | |
Hexamethyldisilazane | Sigma-Aldrich | 440191 | Reagent grade, ≥99% |
Incubator for bioreactor | Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany | ||
Live/Dead Cell Double Staining Kit | Fluka | 04511KT-F | |
Magnetic stirrer plate | 2Mag | 80002 | |
Medium 199 | Sigma-Aldrich | M0650 | 10X |
Microplate reader Tecan Infinite M200 |
Tecan | ||
Needle 21G 16mm | VWR | 613-5389 | |
Papain from papaya latex | Sigma-Aldrich | P4762 | lyophilized powder, ≥ 10 units/mg protein |
Paraffin | Carl Roth GmbH | 6642.6 | |
Penicillin/Streptomycin | Sigma-Aldrich | P4333 | |
Peristaltic pump | Ismatec | ||
Quanti-iT PicoGreen dsDNA assay kit | Thermo Fischer Scientific | P7589 | |
Histofix 4% | Carl Roth GmbH | P087 | |
Scanning Electron Microscope Supra 25 | Carl Zeiss AG | ||
Sodium hydroxide solution 1.0 N | Sigma-Aldrich | S2770 | |
Spinner flasks (25 mL) | Wheaton | 356879 | |
Syringe 1 mL | VWR | 720-2561 | |
Tissue culture flasks (25 cm2, 75 cm2, 150 cm2) | TPP Techno Plastik Products AG | ||
Trypan blue 0.4% | Sigma-Aldrich | T8154 | |
VascuLife VEGF-Mv | Lifeline cell technology | LL-0005 |