Large-scale expansion of Wharton’s jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing

Abstract

IntroductionSuccessful stem cell therapy relies on large-scale generation of stem cells and their maintenance in a proliferative multipotent state. This study aimed to establish a three-dimension culture system for large-scale generation of hWJ-MSC and investigated the self-renewal activity, genomic stability and multi-lineage differentiation potential of such hWJ-MSC in enhancing skin wound healing.MethodshWJ-MSC were seeded on gelatin microbeads and cultured in spinning bottles (3D). Cell proliferation, karyotype analysis, surface marker expression, multipotent differentiation (adipogenic, chondrogenic, and osteogenic potentials), and expression of core transcription factors (OCT4, SOX2, NANOG, and C-MYC), as well as their efficacy in accelerating skin wound healing, were investigated and compared with those of hWJ-MSC derived from plate cultres (2D), using in vivo and in vitro experiments.ResultshWJ-MSC attached to and proliferated on gelatin microbeads in 3D cultures reaching a maximum of 1.1–1.30 × 107cells on 0.5 g of microbeads by days 8–14; in contrast, hWJ-MSC derived from 2D cultures reached a maximum of 6.5 -11.5 × 105 cells per well in a 24-well plate by days 6–10. hWJ-MSC derived by 3D culture incorporated significantly more EdU (P < 0.05) and had a significantly higher proliferation index (P < 0.05) than those derived from 2D culture. Immunofluorescence staining, real-time PCR, flow cytometry analysis, and multipotency assays showed that hWJ-MSC derived from 3D culture retained MSC surface markers and multipotency potential similar to 2D culture-derived cells. 3D culture-derived hWJ-MSC also retained the expression of core transcription factors at levels comparable to their 2D culture counterparts. Direct injection of hWJ-MSC derived from 3D or 2D cultures into animals exhibited similar efficacy in enhancing skin wound healing.ConclusionsThus, hWJ-MSC can be expanded markedly in gelatin microbeads, while retaining MSC surface marker expression, multipotent differential potential, and expression of core transcription factors. These cells also efficiently enhanced skin wound healing in vivo, in a manner comparable to that of hWJ-MSC obtained from 2D culture.

Scalable Manufacturing of Human Mesenchymal Stromal Cells in the Vertical‐Wheel Bioreactor System: An Experimental and Economic Approach

“…Importantly, the process did not compromise UCM identity, since they maintained the typical MSC immunophenotype and multilineage differentiation potential . To our best knowledge, the expansion results obtained are comparable, or even superior, to those described in the literature for MSC obtained from UCM …”

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells

“…Although an initial lag phase was observed for the first 2 days of culture, a maximal cell density of 3.5 × 10 5 ± 1.0 × 10 5 cells/ml was obtained after 5.5 days of culture, representing a total cell FI of 13 ± 2.5 (Figure a,b). These expansion results are comparable to, or even superior to, those described in the literature using CultiSpher‐G (animal gelatin‐origin) (Zhao et al , ) or Cytodex 1 (dextran‐based) (Hupfeld et al , ) microcarriers. Throughout the 6 days of culture, cell viability was measured and was always above 90%.…”

“…Considering that the same set of experimental conditions was tested for MSC from the bone marrow, adipose tissue, synovial tissue and UCM, this suggests that intrinsic features of MSCs from different sources should be considered when developing strategies for large‐scale MSC manufacturing. Indeed, others have used stirred microcarrier‐based culture systems for the expansion of UCM MSCs, although using poorly defined xenogeneic components, such as FBS as a culture supplement (Hupfeld et al , ; Teixeira et al , ; Zhao et al , ) or animal‐derived microcarriers (Zhao et al , ) making GMP compliance difficult. In this context, taking into account the high proliferative potential of UCM MSCs cultured in DMEM + 5% UltraGRO TM under static conditions and that cells retained their immunophenotype and multilineage differentiation potential after five consecutive passages, the use of this HPL‐based product was exploited as a culture component to expand these cells in the previously established plastic microcarrier‐based culture system (dos Santos et al , ; Carmelo et al , ).…”

The survival of transplanted mesenchymal stem cells from humans Wharton’s jelly of the umbilical cord in the central nervous system of rats with experimental allergic encephalomyelitis after their suboccipital injection

“…МСК з ВДл можуть бути ефективними в лікуванні захворювань ЦНС. Імуномодулюючий ефект МСК ВДл оцінений in vitro і на моделях на тваринах [15]. Вважають, що ЦНС є забар'єрним імуно-привілейованим органом завдяки її структурним особливостям.…”