Isolation and cultivation of adipose tissue-derived mesenchymal stromal cells

Commonly used acronym: AT-MSC

Scope of the method

Alternative method relates to
  • Animal health
Alternative method is situated in
  • Basic Research
Type of alternative method
  • In vitro - Ex vivo
This method makes use of
  • Human derived cells / tissues / organs
Specify the type of cells/tissues/organs
adipose tissue-derived mesenchymal stromal cells

Description

Method keywords
  • adipose tissue
  • Stem cells
  • mesenchymal stromal cells
  • isolation
  • cultivation
Scientific area keywords
  • stem cell culture
  • stem cell isolation
  • mesenchymal stromal cells
Method description

Approximately 125 g of processed adipose tissue is incubated for 90 minutes at 37°C in dissociation medium (1:1) consisting of 1% (v/v) bovine serum albumin and 1 mg/mL collagenase A in phosphate buffered saline (PBS). After two filtration steps, the filtrate is carefully brought on top of 15 mL of Histopaque®-1077. Upon centrifugation for 20 minutes at 1000 g (4°C), the top layer is removed and the AT-MSC are collected in 50 mL PBS/BSA (1%). This procedure is carried out separately on two pieces of adipose tissue. Typically 5 - 20 x 10E7 viable cells are obtained per 250 g of processed adipose tissue. The isolated AT-MSC are then (sub)cultured as a monolayer in AT-MSC growth medium for 2 weeks, consisting of Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) foetal bovine serum (FBS), 50 µg/mL streptomycin sulphate, 7.33 IU/mL benzyl penicillin and 2.5 µg/mL fungizone. Cell cultures are incubated at 37°C in a 5% (v/v) CO2 humidified atmosphere and passaged at subconfluency using TrypLE® express. Growth media is changed every 3 days.

Lab equipment
  • Biosafety cabinet level 2 ;
  • Cell incubator ;
  • Centrifuge.
Method status
  • History of use
  • Internally validated
  • Published in peer reviewed journal

Pros, cons & Future potential

Advantages

Robust isolation method for adipose tissue-derived mesenchymal stromal cells.

References, associated documents and other information

References

De Kock J, Najar M, Bolleyn J, Al Battah F, Rodrigues RM, Buyl K, Raicevic G, Govaere O, Branson S, Meganathan K, Gaspar JA, Roskams T, Sachinidis A, Lagneaux L, Vanhaecke T, Rogiers V. (2012) Mesoderm-derived stem cells: the link between the transcriptome and their differentiation potential. Stem Cells Dev. 21(18):3309-23

Najar M, Rodrigues RM, Buyl K, Branson S, Vanhaecke T, Lagneaux L, Rogiers V, De Kock J. (2014) Proliferative and phenotypical characteristics of human adipose tissue-derived stem cells: comparison of Ficoll gradient centrifugation and red blood cell lysis buffer treatment purification methods. Cytotherapy. 16(9):1220-8

Associated documents

Contact person

Joery De Kock

Organisations

Vrije Universiteit Brussel
Pharmaceutical and Pharmacological Sciences (FARM)
In Vitro Toxicology and Dermato-cosmetology
Belgium
Brussels Region