Large-Scale Expansion of Human Mesenchymal Stem Cells

Table 1

Summary of the articles describing the large-scale expansion of MSCs.

Reference	Bioprocessing method	Cell source	Cell culture medium	Initial cell seeding	Culture period (days)	Final cell yield	Expansion ratio	Doubling time (h)	MSC characterization	Other key findings
[6]	CellSTACK 2-chamber	WJ-MSCs	DMEM-KO with 10% FBS and 2 ng/ml bFGF		5		195.28	15.77	(i) Positive for CD44, CD73, CD90, CD105, CD146, and CD166; (ii) Negative for CD34, CD45, and HLA-DR; (iii) Maintained the trilineage differentiation potential; (iv) Maintained the capability to inhibit T-cell proliferation; (v) No chromosome abnormality; (vi) No hTERT expression; (vii) No changes in expression of p53, p21, p16, and c-Myc	(i) Presence of bFGF boosted the cell growth
[17]	CellSTACK 5-chamber	DP-MSCs	DMEM-KO with 10% HPL		11		156.60	36.21	(i) >80% positivity for CD44, CD73, and CD90; (ii) <2% positivity for CD34, CD45, and HLA-DR; (iii) Maintained the trilineage differentiation potential; (iv) Maintained the expression of stem cell markers, i.e., SOX2, OCT4, and NANOG	(i) Supplementation with HPL gave higher cell yield
			DMEM-KO with 10% FBS				100.63	39.68
[22]	Cell Factory 4-chamber	BM-MSCs	αMEM with 10% HPL and 2 IU/ml heparin	(BM-MNCs)	13.5		4.11	159.02	(i) >95% positivity for CD73, CD90, and CD105; (ii) <2% positivity for HLA-DR, CD14, CD19, CD34, and CD45; (iii) Maintained the trilineage differentiation potential
[23]	CellSTACK 2-chamber	BM-MSCs	αMEM supplemented with 8% HPL and 1 IU/ml heparin		7		10.37	49.79	(i) >95% positivity for CD73, CD90, CD105, and HLA-ABC; (ii) <1% positivity for CD3, CD34, and CD45; (iii) Maintained the trilineage differentiation potential	(i) Yield decreased for MSCs cultured with 10% HPL for 7 days likely due to cell detachment caused by culture over confluence
			αMEM supplemented with 8% HPL and 1 IU/ml heparin		5		9.22	37.44
			αMEM supplemented with 10% HPL and 1 IU/ml heparin		7		7.15	59.21
			αMEM supplemented with 10% HPL and 1 IU/ml heparin		5		11.10	34.56
[36]	Hyperflask	VC-MSCs	DMEM/F-12 with 10% FBS and 3 ng/ml bFGF		4		14.26	23.75	(i) >95% positivity for CD73, CD90, and CD105; (ii) <1% positivity for CD34, CD45, CD79, and HLA-DR; (iii) Maintained the trilineage differentiation potential; (iv) Normal karyotype
[37]	Cell Factory 4-chamber	BM-MSCs	αMEM with 10% FBS		15		316.25	49.80	(i) Maintained the trilineage differentiation potential
[7]	Spinner flask+Cytodex 3 microcarrier	BM-MSCs	αMEM with 15% FBS		7		3.86	86.28	(i) >90% positivity for CD73, CD90, and CD105; (ii) Maintained the trilineage differentiation potential; (iii) No chromosome abnormality; (iv) Maintained the capability to inhibit lymphocyte proliferation
[16]	Spinning bottle+CultiSpher-G microcarrier	WJ-MSCs	MEM/F12 with 10% FBS and 10 ng/ml bFGF		6		2.60	104.46	(i) >95% positivity for CD44, CD73, CD90, and CD105; (ii) Low expression of CD31 and CD45; (iii) No chromosomal abnormality; (iv) Maintained the trilineage differentiation potential; (v) High expression of embryonic markers (OCT4, SOX2, NANOG, and C-MYC)	(i) The cells expanded with spinner flask were more efficient in promoting in vivo wound healing compared to those expanded with culture flask
[18]	Spinner flask+plastic microcarrier	BM-MSCs	DMEM with 10% FBS		6		2.86	94.99	(i) >99% positivity for CD73, CD90, and CD105; (ii) <1% positivity for HLA-DR; (iii) Maintained the trilineage differentiation potential	(i) Serum-free medium enhanced cell growth
	Spinner flask+plastic microcarrier coated with fibronectin		PRIME-XV™ SFM				10.03	43.29
[24]	Spinner flask+Cultispher S microcarrier	PD-MSCs	DMEM-HG with 10% FBS		10		2.7	167.5	(i) Simultaneous expression of CD73, CD90, and CD105 in 89% of MSCs cultured with FBS and 86% in those cultured with HPL; (ii) Low expression of negative markers for both mediums; (iii) Sox9, ALP, BMP2, and WNT5A were upregulated in MSCs cultured with HPL compared to those cultured with FBS; (iv) In vivo study showed that MSCs cultured with HPL formed more matured mineralization tissue compared to those cultured with FBS which form fibrous tissue	(i) Shear stress affected expression of percentage of positive surface marker; (ii) MSCs cultured with HPL are more potent in bone formation in vivo
			DMEM-HG with 10% HPL		10		5.2	100.9
[25]	Spinner flask+plastic microcarrier	WJ-MSCs	DMEM-LG with 5% UltraGRO™ and 2 IU/ml heparin		5.5		7.00	47.02	(i) >95% positivity for CD73, CD90, and CD105; (ii) Maintained the trilineage differentiation potential
[29]	Spinner flask+Corning Synthemax II microcarrier	BM-MSCs	Mesencult™-XF		7		5.00	72.35	(i) >95% positivity for CD73 and CD105; (ii) <1% positivity for CD14 and CD45; (iii) Normal karyotype; (iv) Maintained the trilineage differentiation potential	(i) Culture with Stemgro hMSC gave higher cell yield and expansion ratio and lower population doubling time
			Stemgro hMSC				7.00	59.84
[30]	Spinner flask+microcarrier	BM-MSCs	StemPro MSC SFM Xenofree		8		7.20	67.42	(i) Cells expressed high level of CD105, CD73, and CD90 and lower level of CD31, CD80, and HLA-DR; (ii) Maintained the trilineage differentiation potential
		AT-MSCs					10.13	57.80
[31]	Spinner flask+Cultispher® S microcarrier coated with CELLstart CTS solution	WJ-MSCs	StemPro MSC SFM XenoFree		5		4.80	53.03	(i) Maintain expression of CD90 and CD73 postexpansion; (ii) Expression of CD105 decreased postexpansion, probably due to cell damage by shear stress; (iii) Low expression of CD31, CD80, and HLA-DR; (iv) Maintained the trilineage differentiation potential; (v) Maintained the capability to inhibit lymphocyte proliferation	(i) Cells cultured with bioreactor have higher expansion ratio compared to those expanded using the spinner flask
	2.5 l Celligen 310 bioreactor+Cultispher® S microcarrier coated with CELLstart CTS solution				4		5.60	38.63
[33]	Spinner flask+plastic microcarrier coated with CELLstart CTS solution	BM-MSCs	StemPro MSC SFM XenoFree		14		4.00	168.00	(i) >95% positivity for CD73 and CD105; (ii) <2% positivity for CD31, CD80, and HLA-DR; (iii) 92% and 82% CD90 positivity for BM-MSCs and AT-MSCs, respectively, likely due to damage to the cells caused by longer enzymatic cell detachment process or shear stress; (iv) Maintained the trilineage differentiation potential
		AT-MSCs					2.80	226.20
[56]	Spinner flask+Cytodex 3 microcarrier	F-MSCs	αMEM with 10% FBS		7		8.85	53.40	(i) >95% positivity for CD73, CD90, and CD105; (ii) <1% positivity for CD34	(i) Cells cultured with spinner flask have better osteogenic differentiation potential compared to those cultured in culture flask
[19]	Quantum Cell Expansion System	AT-MSCs	αMEM with 10% FBS		21		5.67	201.4	(i) >95% positivity for CD90, CD73, CD105, CD13, CD166, and CD29; (ii) <5% positivity for CD45, CD19, CD31, and HLA-DR; (iii) No genomic instability; (iv) Maintained the trilineage differentiation potential	(i) HPL is superior compared to FBS; (ii) 1.66-fold to 8.15-fold of AT-MSCs was harvested from SVF seeded at P0
			αMEM with 5% heparin-free HPL		6		28.81	29.70
[20]	Stirred tank reactor	AT-MSCs	αMEM with 10% HPL and 1 IU/ml heparin in 21% O2		6		1.85	162.80	(i) Positive for CD73, CD90, and CD105; (ii) Negative for CD14, CD20, CD35, CD45, and HLA-DR; (iii) Maintained the trilineage differentiation potential	(i) Hypoxic cells displayed slightly poorer osteogenic differentiation potential and slightly better adipogenic and chondrogenic differentiation potential
			αMEM with 10% HPL and 1 IU/ml heparin in 5% O2				2.23	124.40
[21]	Mobius® 50 l bioreactor+collagen-coated microcarrier	BM-MSCs	αMEM supplemented with 5% HPL and 2 IU/ml heparin		11		42.67	48.75	(i) >95% positivity for CD105, CD90, CD73, and CD44; (ii) <5% positivity for CD19, CD34, CD 11b, CD79a, CD45, and CD14; (iii) Low expression of HLA-DR; (iv) Maintained the trilineage differentiation potential; (v) Maintained the immunosuppressive properties
[27]	Vertical Wheel bioreactor+Synthemax II microcarrier	BM-MSCs	MesenCult-XF with 0.025% () antifoam C emulsion		14		12.00	93.72	(i) Positive for CD44, CD73, CD90, CD105, and CD166; (ii) Negative for CD34 and CD45; (iii) Low expression of HLA-DR; (iv) Maintained the trilineage differentiation potential	(i) Cells cultured with Vertical Wheel bioreactor have significantly lower expression of HLA-DR compared to those cultured with Biostat Qplus bioreactor
	Biostat Qplus bioreactor+Synthemax II microcarrier						11.00	97.10
[28]	2 l UniVessel® SU bioreactor+Synthemax® II microcarrier	BM-MSCs	Mesencult™-XF		7		16.88	41.20	(i) >95% positivity for CD44, CD73, and CD90; (ii) <5% positivity for CD45, CD34, CD14, CD19, and CD11b; (iii) Positivity of CD105 was 88% and 92%, respectively, for BM-MSCs and AT-MSCs; (iv) Maintained the trilineage differentiation potential
		AT-MSCs					20.24	38.72
[32]	1.3 l BioFlo 110 bioreactor+plastic microcarrier coated with CELLstart CTS solution	BM-MSCs	StemPro MSC SFM XenoFree		7		22.00	21.59	(i) >90% positivity for CD73; (ii) Expression of CD90 and CD105 decreased to 74% and 39%, respectively, for BM-MSCs; (iii) Expression of CD90 dropped to 64% (from graph) for AT-MSCs; (iv) <2% positivity for CD31, CD80, and HLA-DR
		AT-MSCs					9.00	25.88
[35]	2.5 l Celligen 310 bioreactor+Fibra-Cel® disk	BM-MSCs	αMEM with 10% FBS		9		9.20	67.47	(i) >90% positivity for CD44, CD90, and CD105; (ii) Maintained the trilineage differentiation potential
[38]	Quantum Cell Expansion System bioreactor	WJ-MSCs	F12K: DMEM-LG (1 : 1) with 10% FBS		7		19	39.5	(i) >99% positivity for CD44, CD73, CD90, and CD105; (ii) <1% positivity for CD45, CD34, CD11b, CD19, and HLA-DR; (iii) MSCs maintained the trilineage differentiation potential; (iv) Maintained the capability to inhibit lymphocyte proliferation; (v) No alteration in karyotype	(i) There was no difference in cell proliferation and growth properties between MSCs cultured in flask and bioreactor
[39]	Pall Life Sciences Xpansion Multiplate Bioreactor	PD-MSCs	DMEM-HG with 10% FBS		7		3.34	96.47	(i) >90% positivity for CD73, CD90, and CD105; (ii) <5% positivity for CD45, CD20, CD14, and CD34; (iii) Maintained the trilineage differentiation potential	(i) 55% cell lost during the downstream process
[26]	HYPERFlasks®	WJ-MSCs	αMEM with 15% AB human serum		11		12.99	71.36	(i) Positive for CD73, CD90, and CD105; (ii) Negative for CD12, CD31, CD34, CD45, and HLA-DR; (iii) Cells cultured with HYPERFlasks® showed reduction in CD73 expression; (iv) Cells cultured with bioreactor showed reduction in CD105 expression, likely due to cell damage by shear stress; (v) Maintained the trilineage differentiation potential; (vi) Maintained the chromosome stability; (vii) Maintained the immunosuppressive properties
	Roller bottle				6		7.01	68.45
	Spinner flask+plastic microcarrier				8		21.00	32.78
	2.5 l Celligen 310 bioreactor+plastic microcarrier				7		9.90	50.79

MSCs: mesenchymal stem cells; BM-MSCs: bone marrow-derived MSCs: BMNCs: bone marrow mononuclear cells; WJ-MSCs: Wharton’s jelly-derived MSCs; AT-MSCs: adipose tissue-derived MSCs; PD-MSCs: periosteum-derived MSCs; VC-MSCs: villous chorion-derived MSCs; DP-MSCs: dental pulp-derived MSCs; F-MSCs: fetal MSCs; HPL: human platelet lysate: FBS: fetal bovine serum; bFGF: basic fibroblast growth factor.