Toward a Clinical-Grade Expansion of Mesenchymal Stem Cells from Human Sources: A Microcarrier-Based Culture System Under Xeno-Free Conditions

Abstract

The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0±0.2)×10⁵ cells·mL⁻¹ (18±1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4±0.5)×10⁵ cells·mL⁻¹ (14±7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practices-compliant large-scale production system of MSC for cellular therapy.

Suspension-Expansion of Bone Marrow Results in Small Mesenchymal Stem Cells Exhibiting Increased Transpulmonary Passage Following Intravenous Administration

“…High yield MSC expansion on similar microbead carriers has been previously demonstrated for both rat 23,24 and human cells. 25,26 Unlike these prior reports, we expanded these cultures by adding media and microbead carriers, while retaining the nonadherent cellular fractions. The rationale behind this approach is to allow for the retention of Green fluorescent cells were isolated by plating from bioreactor MSCs injected rat arterial blood, but not from the monolayer MSCs experiments (inset).…”

Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor

“…Cultispher-S (Eibes et al 2010), Cultispher-G (Sun et al 2010) and SoloHill Plastic P102-L (Dos Santos et al 2011). However in all the aforementioned studies, the largest volume used was no more than 200 mL and it is unclear as to whether samples of microcarrier suspension greater than 2 mL were harvested.…”

“…These microcarriers were selected based on their suitability for the culture of hMSCs relative to a number of other microcarriers as identified in previous unpublished studies conducted by the authors (data not shown) as well as other groups (Dos Santos et al 2011), in addition to their amenability for good manufacturing practise (GMP) bioprocesses, given their xeno-free composition. Whilst it would be possible to control the pH and dO 2 levels in the bioreactors, it was decided that this investigation would be used to monitor and identify the change in pH and dO 2 over the course of the culture as there is little work in the literature which identifies the optimal pH and dO 2 levels for microcarrier hMSC culture.…”

Application of Bioreactor in Stem Cell Culture

“…Thus, serum free culture is a direction in the future standardized production of biological products with bioreactors [51,52]. It is necessary to investigate the metabolic characteristics of SCs in large scale expansion of cells, design the key components in the serum free medium and optimize the physical and chemical properties of the medium, which may develop individualized culture medium [53]. This, together with appropriate fed-batch or perfusion strategies, may assure the key nutrients at appropriate levels, which reduces the production of toxins and is suitable for large-scale cell expansion [10,54].…”