Sampling and isolation experiments of adipose-derived stem cells

Summary

Stem and progenitor cell populations are present in a variety of adult tissues, including skin, muscle, bone marrow, and fat. A growing body of research suggests that these cells may have the potential for multilineage differentiation, capable of generating cell types other than the tissue of origin. In recent years, it has been discovered that adult adipose tissue can be an additional rich source of MSCs. This residual cell population has been named adipose-derived stem cells (ASCs). Under specific culture conditions, ASCs can be induced to differentiate into a variety of mesenchymal and neural cell types. Source: Stem Cell Culture

Operation method

Acquisition of ASCs from mouse inguinal fat pads

Principle

Both human and mouse adipose tissue can be used to isolate ASCs, and a fair number of ASCs can be obtained. human adipose tissue is usually derived from liposuction. Mouse adipose tissue is obtained from the inguinal fat pad.

Materials and Instruments

Mice 4~6 mice
Hank's buffered salt solution (HBSS) ASC medium Collagenase solution Polyvinylpyrrolidone iodine solution Anesthetic: tribromoethanol 37°C water bath
Petris Petri dishes 9 cm centrifuge tubes 50 ml tissue culture flasks 25 cm2 Scissors (2) Tweezers (2)

Move

(a) HBSS and ASC media were pre-warmed in a 37°C water bath.

(b) Anesthetize the animals and execute them.

(c) Transferring animals to a laminar flow decontamination hood.

(d) Abdomen was sterilized with 70% alcohol.

(e) The abdominal cavity was opened using a low transverse incision.

(f) Remove the gonads/epidymis and fat pads in the groin with forceps.

(g) Place the fat in a petri dish and add HBSS.

(h)After all animal fat was removed, the fat was transferred to a new petri dish.

(i)Add HBSS containing 5% polyvinylpyrrolidone iodide for 2~3 min.

(j)Rinse the tissue and wash the fat with HBSS.

(k) Transfer the fat to a 50 ml centrifuge tube using sterile forceps.

(l) Chop the fat with sterile scissors.

(m) Collagenase solution of the same volume as the tissue was added and mixed well.

(n) The centrifuge tube was placed in a 37°C water bath for 60 min, during which time it was mixed from time to time.

(o) Centrifuge at 50 to 100 g for 5 min.

(p)Remove the centrifuge tube, shake vigorously (to completely separate the stromal cells from the primary adipocytes), and centrifuge again for 5 min. .

(q)Carefully aspirate the upper layer of grease, which contains primary adipocytes, taking care not to damage the stromal-vascular cell layer located below.

(r) Add 5~10 ml PBSA, resuspend the precipitate, and centrifuge again for 5 min.

(s)Wash three times, taking care not to aspirate the stromal-vascular cell layer.

(t)After the last washing, add 8 ml ASC medium to resuspend the precipitate, transfer to a T25 culture flask, and place in a 37℃, 5% _CO2 incubator.

(u)Allow the cells to grow on the wall for 2~4 days and then change the solution.

(v)Remove the non-adherent cells by discarding the culture medium when changing the solution.

(w)Change the medium twice a week thereafter.

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