Optimization of isolation and culture conditions of highly homogenous human adipose-derived mesenchymal stem cells

Xufang YANG; Xu HE; Lihong ZHANG; Mei SUN; Hong XU; Yunhe GAO; Yulin LI

ABSTRACT

BACKGROUND:

Up to date,there is not an acceptable method for isolating,culture and amplifying human adipose-derived mesenchymal stem cells (hADSCs).

OBJECTIVE:

To explore the most effective way to obtain highly homogenous and undifferentiated hADSCs.DESIGN,TIME AND

SETTING:

The in vitro cytology experiment was performed at the Key Laboratory of Pathobiology,Ministry of Education,Jilin University from June to December 2008.MATERIALSHuman abdominal adipose tissue resected in the surgery was supplied by the Affiliated Hospital of Jilin University,The informed consent was obtained from patients.

METHODS:

Human adipose tissue was removed connective tissue and blood vessel,followed by incubation in 0.1% type I collagenase for 60 minutes at 37℃,filtrated then centrifuged.Consequently,the subnatant precipitation was cultured with LG-DMEM containing 10% fetal bovine serum,incubated at 6-well plate with density of 1×10~9/L,and placed in incubator of 5% CO_2 at 37 ℃.The cultured cells were passaged when the cells reached 80%-90% confiuency,and the 3~(rd) passage of cells were induced to osteogenic and adipogenic differentiation.MAIN OUTCOME

MEASURES:

Morphological characteristics of hADSCs were observed by laser scanning confocal microscope.Immunophenotypes,cell cycle and growth curve of hADSCs were detected by flow cytometry and immunofiuorescent techniques.In addition,the multiple differentiation potential of hADSCs was detected.

RESULTS:

hADSCs presented fibroblast-like morphological feature with a flocked array.The 3~(rd) passage of hADSCs had unique immunophenotypes and they were positive for CD73,CD44,CD166,CD105 and CD29,but negative for CD31,CD34,CD45 and HLA-DR.Cell cycle result showed that they had the typical growth characteristics of stem calls,namely,83.81% cells stayed at G_0/G_1 stage,only 16.19% cells were stayed at S+G_2/M stage;The latent phase of the primary culture cells was 2 days prior to and after incubation,followed by 3-6 days of logarithmical proliferation,reached a peak at day 6,and entering the growth platform phase with lower growth speed.The alkaline phosphatase was positive expressed at week 2 of osteogenic induction.And the positive expression of oil-O red staining could be seen at day 3 of adipogenic induction.

CONCLUSION:

Cells contamination can be reduced by removing connective tissue and blood vessel of adipose tissue,and 0.1% type Ⅰ collagenase for 60 minutes can effective separated stroma cell to matrix fiber,furthermore,ensure a sufficient contact between collagenase and tissures,which provide an supportive for harvesting highly homogenous hADSCs.