Defined serum- and xeno-free cryopreservation of mesenchymal stem cells

Al-Saqi, Shahla Hamza; Saliem, Mohammed; Quezada, Hernán Concha; Ekblad, Åsa; Jonasson, Aino Fianu; Hovatta, Outi; Götherström, Cecilia

doi:10.1007/s10561-014-9463-8

Cited by 34 publications

(20 citation statements)

References 139 publications

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“…3,33,34 In contrast, other studies 10,[35][36][37] have demonstrated that cryopreserved MSC exhibit equivalent viability and immunosuppressive potential to freshly isolated MSC from cell culture. Consistent with these findings, we found that freeze-thawed MEP displayed a viability of 95% and retained the ability to effectively suppress lymphocyte proliferation in vitro.…”

Section: Discussionmentioning

confidence: 95%

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

et al. 2016

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© Ferrata Storti Foundation IntroductionSince the first clinical trial of mesenchymal stromal cells (MSC) 9,10 Another important issue regarding the clinical application of MSCs is their culture under serum-free conditions. The majority of clinical studies have used MSCs that were expanded in media supplemented with fetal bovine serum (FBS). 1,[11][12][13][14][15] To avoid the risks associated with the use of FBS, 16 platelet lysate (PL) was proposed as a supplement to tissue culture media for MSCs. 17 Recently, several studies showed that MSCs that were expanded in PL exhibited the same efficacy as MSCs cultured in serum-containing media for the treatment of GvHD. 18-22To date, clinical studies have used MSCs that have been generated from several individual donors. Considering the aforementioned inter-donor heterogeneity and the need for a large number of "off-the-shelf" MSCs, the establishment of MSC banks appears to be an indispensable strategy for providing a continuous supply of MSCs with predictable potency. To our knowledge, there are few established MSC banks worldwide, and these MSC banks were generated by separately isolating, expanding, and freezing MSCs from up to 10 donors in FBS-containing media. [23][24][25][26] In the current study, we report for the first time the establishment of a serum-free and GMP-compliant MSC bank generated from pooled bone marrow mononuclear cells (BM-MNCs) of multiple donors as a novel strategy to circumvent donor-to-donor variability. Clinical-grade MSC endproducts (MEPs) derived from the MSC bank were thoroughly assessed for their proliferation, differentiation, and, in particular, for the allosuppressive potential in vitro. Importantly, 81 MEPs were administered as a rescue therapy to 26 pediatric patients with severe steroid-refractory aGvHD in seven transplantation centers. Safety and efficacy of MEPs was compared to MSCs generated from a single or several individual donors that have been used in the GvHDclinical studies reported thus far. Methods Generation of MSC bank and clinical-grade MEPsBone marrow was collected from 8 healthy volunteers (age 21-45 years old) after written informed consent and after the approval of the local Ethics Committee (n. 275/09). BM-MNCs were enriched from the bone marrow aspirate by using the Sepax II NeatCell process (Biosafe, Eysins, Switzerland) and frozen individually. After thawing and washing these BM-MNCs were pooled. This pool of BM-MNCs from 8 donors was used to generate MSCs over 14 days in culture. After their detachment, passage 1 mesenchymal stromal cells (MSC-P1) were washed and aliquoted into 209 cryovials (each containing 1.5x10 6 MSC-P1). Cryopreserved vials with MSC-P1 were referred to as the MSC bank.To generate clinical-grade MEPs, MSC-P1 aliquots from the MSC bank were thawed and after washing they were expanded in medium containing 10% PL till the end of passage 2. These MSCs were re-suspended in cryomedium (0.9% NaCl containing 5% HSA and 10% DMSO), distributed in cryobags (each containing 1-3x10 6 MS...

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Section: Discussionmentioning

confidence: 95%

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

et al. 2016

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“…The list of commercially available chemically defined, protein-free, xeno-free and serum-free is constantly increasing [29] as discussed in recent reviews [30,31].…”

Section: Stem Cell Mediamentioning

confidence: 99%

“…To improve outcome of cryopreservation, the authors suggested nucleation temperature control during the freezing process [108]. Another recent report described the formulation of hMSCs using the FDA-approved commercial serum-free and xeno-free CPA known as STEM-CELLBANKER™ (Amsbio, UK) [29]. The cell viability was >90% (better than DMSO) and no morphological differences were observed, but cell growth was slower.…”

Section: Formulation and Storage Of Stem Cellsmentioning

confidence: 99%

“…The cell viability was >90% (better than DMSO) and no morphological differences were observed, but cell growth was slower. The mesodermal differentiation capacity was not regardless of which CPA was used [29]. The cryopreservation of hMSCs has also been tested using Prime-XV MSC FreezIS DMSO-Free (Irvine Scientific, USA) with 30 min incubation at room temperature before cooling to 4°C for 5 min followed by further cooling at 1°C/ min down to -80°C for storage in liquid nitrogen vapor.…”

Section: Formulation and Storage Of Stem Cellsmentioning

confidence: 99%

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The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

Elseberg¹,

Leber²,

Weidner³

et al. 2017

New Insights Into Cell Culture Technology

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In the field of cell therapy, allogenic human mesenchymal stromal cells (hMSCs) are often used in clinical trials, creating a demand for cell mass production using efficient dynamic bioreactor systems. As an advanced therapy medicinal product (ATMP), such cells should meet certain special requirements, including product specifications requiring a production process compatible with good manufacturing practice (GMP). The development of processes in which the cells are the product therefore remains a significant challenge. This chapter describes the requirements at different steps in the upstream and downstream phases of such dynamic processes. Potential solutions are presented and future prospects are discussed, including the selection of media and carriers for the strictly adherent growing cells, allowing efficient cell adhesion and detachment. Strategies for dynamic cultivation in bioreactors are described in detail for fixed-bed and stirred-tank reactors based on GMP requirements and the integration of process analytical technology (PAT). Following cell harvest, separation and purification, the formulation and storage of the product are also described. Finally, the chapter covers important cell quality characteristics necessary for the approval of ATMPs.

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“…50,51 To assess the osteogenic potential of cryopreserved MSCs, the presence of calcium deposition after osteogenic induction of cryopreserved MSCs was confirmed by Alizarin Red or Von Kossa staining. 10,13,51,53,54 These osteogenic-like cells showed a high expression level of osteogenic markers such as osteocalcin (OSC) and alkaline phosphatase (ALP). 51,54 The presence of proteoglycan after chondrogenic induction of cryopreserved MSCs was confirmed by Alcian Blue or Safranin O staining.…”

Section: Cell Differentiation Capacitymentioning

confidence: 99%

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

Yong

Zaman

Xufeng

et al. 2015

Biopreservation and Biobanking

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Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

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Defined serum- and xeno-free cryopreservation of mesenchymal stem cells

Cited by 34 publications

References 139 publications

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey

The Challenge of Human Mesenchymal Stromal Cell Expansion: Current and Prospective Answers

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges

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