Dataset Information

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.

ABSTRACT: Umbilical cord derived mesenchymal stromal cells (UC-MSCs) are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP) for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed-vessel dissociation following enzymatic digestion which reduces contamination risk and manipulation time. The new method produced >10 times more cells per cm of UC than our previous method. When biographical variables were compared, more UC-MSCs per gram were isolated after vaginal birth compared to Caesarian-section births, an unexpected result. UC-MSCs were expanded in medium enriched with 2%, 5%, or 10% pooled human platelet lysate (HPL) eliminating the xenogeneic serum components. When the HPL concentrations were compared, media supplemented with 10% HPL had the highest growth rate, smallest cells, and the most viable cells at passage. UC-MSCs grown in 10% HPL had surface marker expression typical of MSCs, high colony forming efficiency, and could undergo trilineage differentiation. The new protocol standardizes manufacturing of UC-MSCs and enables clinical translation.

SUBMITTER: Smith JR

PROVIDER: S-EPMC4757747 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.

Smith J Robert JR Pfeifer Kyle K Petry Florian F Powell Natalie N Delzeit Jennifer J Weiss Mark L ML

Stem cells international 20160204

Umbilical cord derived mesenchymal stromal cells (UC-MSCs) are a focus for clinical translation but standardized methods for isolation and expansion are lacking. Previously we published isolation and expansion methods for UC-MSCs which presented challenges when considering good manufacturing practices (GMP) for clinical translation. Here, a new and more standardized method for isolation and expansion of UC-MSCs is described. The new method eliminates dissection of blood vessels and uses a closed ...[more]

PMID: 26966439

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Project description:BackgroundSafety evaluations in preclinical studies are needed to confirm before translating a cell-based product into clinical application. We previously developed a serum-free, xeno-free, and chemically defined media (S&XFM-CD) for the derivation of clinical-grade umbilical cord-derived MSCs (UCMSCs), and demonstrated that intraperitoneal administration of UCMSCs in S&XFM-CD (UCMSCS&XFM-CD) exhibited better therapeutic effects than UCMSCs in serum-containing media (SCM, UCMSCSCM). However, a comprehensive investigation of the safety of intraperitoneal UCMSCS&XFM-CD treatment should be performed before clinical applications.MethodsIn this study, the toxicity, immunogenicity and biodistribution of intraperitoneally transplanted UCMSCS&XFM-CD were compared with UCMSCSCM in rats via general vital signs, blood routine, blood biochemistry, subsets of T cells, serum cytokines, pathology of vital organs, antibody production and the expression of human-specific gene. The tumorigenicity and tumor-promoting effect of UCMSCS&XFM-CD were compared with UCMSCSCM in nude mice.ResultsWe confirmed that intraperitoneally transplanted UCMSCS&XFM-CD or UCMSCSCM did not cause significant changes in body weight, temperature, systolic blood pressure, diastolic blood pressure, heart rate, blood routine, T lymphocyte subsets, and serum cytokines, and had no obvious histopathology change on experimental rats. UCMSCS&XFM-CD did not produce antibodies, while UCMSCSCM had very high chance of antibody production to bovine serum albumin (80%) and apolipoprotein B-100 (60%). Furthermore, intraperitoneally injected UCMSCS&XFM-CD were less likely to be blocked by the lungs and migrated more easily to the kidneys and colon tissue than UCMSCSCM. In addition, UCMSCS&XFM-CD or UCMSCSCM showed no obvious tumorigenic activity. Finally, UCMSCS&XFM-CD extended the time of tumor formation of KM12SM cells, and decreased tumor incidence than that of UCMSCSCM.ConclusionsTaken together, our data indicate that UCMSCS&XFM-CD display an improved safety performance and are encouraged to use in future clinical trials.

Project description:BackgroundMesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components.Methods and findingsWe established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform.ConclusionsThe five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

Dataset Information

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.

Publications

Standardizing Umbilical Cord Mesenchymal Stromal Cells for Translation to Clinical Use: Selection of GMP-Compliant Medium and a Simplified Isolation Method.

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