Optimization and Scale-up Culture of Human Endometrial Multipotent Mesenchymal Stromal Cells: Potential for Clinical Application

Author(s): Rajaraman G, White J, Tan KS, Ulrich D, Rosamilia A, et al.


We have previously identified and purified multipotent mesenchymal stromal cell (MSC)-like cells in the highly regenerative endometrial lining of the human uterus (eMSC) as CD140b⁺CD146⁺ cells. Due to ease of accessibility with minimal morbidity via biopsy, we are proposing to use eMSC in cell-based therapies; however, culture conditions compliant with Good Manufacturing Practice have not been established for eMSC. The aim of this study was to optimize serum-free and xeno-free culture conditions for expansion of eMSC for potential clinical use. Real-time cell assessment (Xcelligence) and MTS viability assays were used to measure attachment and proliferation of freshly isolated, flow cytometry-sorted CD140b⁺CD146⁺ eMSC cultured in several commercially available and in-house serum-free and xeno-free media in combination with five attachment matrices (fibronectin, collagen, gelatin, laminin, and Cell Start-XF®). Comparisons were made with a standard serum-containing medium, DMEM/F-12/10% fetal bovine serum. Under all conditions examined, eMSC attachment and proliferation was greatest using a fibronectin matrix, with Lonza TP-SF® and our in-house DMEM/SF/FGF2/EGF serum-free xeno-product-containing medium similar to serum-containing medium. Hypoxia increased eMSC proliferation in the DMEM/SF/FGF2/EGF serum-free medium. Culture of eMSC for 7 days on a fibronectin matrix in DMEM/SF/FGF2/EGF serum-free media in 5% O₂ maintained greater numbers of undifferentiated eMSC expressing CD140b, CD146, and W5C5 compared to culture under similar conditions in Lonza TP-SF medium. However, the percentage of cells expressing typical MSC phenotypic markers, CD29, CD44, CD73, and CD105, were similar for both media. EMSC showed greater expansion in 2D compared to 3D culture on fibronectin-coated microbeads using the optimized DMEM/SF/FGF2/EGF medium in 5% O₂. In the optimized 2D culture conditions, eMSC retained CFU activity, multipotency, and MSC surface phenotype, representing the first steps in their preparation for potential clinical use.

Similar Articles

Non-expanded adipose stromal vascular fraction cell therapy for multiple sclerosis

Author(s): Riordan NH, Ichim TE, Min WP, Wang H, Solano F, et al.

Isolation and characterization of multi-potent stem cells from human orbital fat tissues

Author(s): Ho JH, Ma WH, Tseng TC, Chen YF, Chen MH, et al.

Isolation and characterization of canine adipose-derived mesenchymal stem cells

Author(s): Neupane M, Chang CC, Kiupel M,Yuzbasiyan-Gurkan V

Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives

Author(s): Planat-Benard V, Silvestre JS, Cousin B, Andre M, Nibbelink M, et al.

Progenitor-enriched adipose tissue transplantation as rescue for breast implant complications

Author(s): Yoshimura K, Asano Y, Aoi N, Kurita M, Oshima Y, et al.

Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: case report

Author(s): Lendeckel S, Jodicke A, Christophis P, Heidinger K, Wolff J, et al.

Adipose-derived stromal cells: Their identity and uses in clinical trials, an update

Author(s): Casteilla L, Planat-Benard V, Laharrague P,Cousin B

A simple modification of the separation method reduces heterogeneity of adipose-derived stem cells

Author(s): Griesche N, Luttmann W, Luttmann A, Stammermann T, Geiger H, et al.

Characterization of adipose tissue-derived cells isolated with the Celution system

Author(s): Lin K, Matsubara Y, Masuda Y, Togashi K, Ohno T, et al.

Sera of overweight people promote in vitro adipocyte differentiation of bone marrow stromal cells

Author(s): Di Bernardo G, Messina G, Capasso S, Del Gaudio S, Cipollaro M, et al.

Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy

Author(s): Perez-Ilzarbe M, Diez-Campelo M, Aranda P, Tabera S, Lopez T, et al.

Inhibition of mesenchymal stromal cells by pre-activated lymphocytes and their culture media

Author(s): Valencic E, Loganes C, Cesana S, Piscianz E, Gaipa G, et al.

Ex vivo expansion of human mesenchymal stem cells in defined serum-free media

Author(s): Jung S, Panchalingam KM, Rosenberg L,Behie LA

Optimizing proliferation and characterization of multipotent stem cells from porcine adipose tissue

Author(s): Wang KH, Kao AP, Wangchen H, Wang FY, Chang CH, et al.

Intravenous administration of auto serum-expanded autologous mesenchymal stem cells in stroke

Author(s): Honmou O, Houkin K, Matsunaga T, Niitsu Y, Ishiai S, et al.

Expectations and strategies regarding islet transplantation: metabolic data from the GRAGIL 2 trial

Author(s): Badet L, Benhamou PY, Wojtusciszyn A, Baertschiger R, Milliat-Guittard L, et al.

Human CD34/CD90 ASCs are capable of growing as sphere clusters, producing high levels of VEGF and forming capillaries

Author(s): De Francesco F, Tirino V, Desiderio V, Ferraro G, D'Andrea F, et al.