Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Summary

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.

Abstract

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.

Introduction

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 le....

Protocol

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

1. Human bone marrow-derived mesenchymal stromal cells (hBMSCs)
   1. Remove....

Results

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 experiment.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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