An Efficient Method to Obtain Dedifferentiated Fat Cells

Summary

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Abstract

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

Introduction

Cell therapy and tissue engineering are hot topics in the field of regenerative medicine^1-5. While these therapeutic modalities hold great promise, several technical issues currently hamper their clinical use. In this regard, as in the generation of iPS cells, all tissue engineering therapies must produce cells free of external gene transductions in order to maintain patient safety. Accordingly, we were the first group to successfully produce human DFAT cells⁶. Several other research groups have since adopted our method to generate DFAT cells of mammalian origin^7-9, further highlighting the usefulness of our model.

Over the course of several studies, we have found that the quality of the cell culture environment may be modified by adjusting the content of the cell medium. This finding has led to an increase in the success rate of DFAT cell production and improved cell quality; both critical factors in efficiently generating cells for future clinical trials. In this regard, an improved DFAT culture medium (DCM, a medium similar to mesenchymal stem cell medium, which contains recombinant human insulin, serum albumin, L-glutamic acid, several fatty acids, and cholesterol) and a method for DFAT cell generation and proliferation was developed (more information about the contents of DCM is available upon request). Using this method high quality DFAT cells were generated with the ability to differentiate into several cell types including adipogenic, osteogenic, and chondrogenic cells. Altogether, this validated cell culture protocol enhances the quality of DFAT cells and may be quite useful for enhancing clinical applications of cell therapy and tissue engineering.

Protocol

Samples of human subcutaneous fat were obtained from patients undergoing surgery in the Departments of Plastic Surgery, Urology, Pediatric Surgery and Orthopedic Surgery of Nihon University Itabashi Hospital (Tokyo, Japan). The patients gave written informed consent, and the Ethics Committee of Nihon University School of Medicine approved the study.

1. Tissue Preparation

1. Bring the tissue sample from the operating room to the laboratory.
2. Wash 1-2 g of adipose tissue with 5 ml of PBS(-) at room temperature (RT) once, in a 50 ml tube and then place the sample in a 10 cm plastic dish.

2. Collagenase Digestion

1. Remove the sample from the PBS(-), then add 10 ml of sterile 0.1% (w/v) collagenase solution (Collagenase type II) to the dish containing the tissue sample.
2. Mince the tissue with surgical scissors for approximately 15 min, until it reaches a pureed consistency.
3. Subsequently, digest the minced tissue in the 0.1% (w/v) collagenase solution at 37 °C for 30 min (60 rpm) in a 50 ml tube with gentle agitation on a shaker.
4. Filter the digested sample through a 100 µm filter into a 50 ml tube. Next, pass 10 ml of DCM (see Table of Materials) supplemented with 2% FBS through the filter.

3. Cell Isolation

1. Centrifuge the filtered cells at 100 x g for 1 min at RT.
2. At this point, prepare 5 ml of DCM supplemented with 2% FBS in a 50 ml tube.
3. Next, draw up the floating fat cells using a 1,000 µl pipette and add the cells to a 50 ml tube containing medium.
4. Subsequently, shake the 50 ml tube gently to mix the DCM medium with the cells.
5. Then, centrifuge the tube at 100 x g for 1 min at RT.
6. Discard the medium at the bottom of the tube using an 18 G needle and a 20 ml syringe.
7. Next, add 10 ml of DCM supplemented with 2% FBS to the collected cells.
8. Mix the DCM with the cells by shaking the 50 ml tube gently.
9. Centrifuge the tube at 100 x g for 1 min at RT.

4. Plating Cells

1. Add 41 ml of DCM or DMEM supplemented with 20% FBS to a 12.5 cm flask.
2. Next, using a 200 µl pipette add 40 µl of fat cells to the flask.
3. Then, fill up the flask with DCM or DMEM supplemented with 20% FBS. CRITICAL STEP: At this point, ensure that the cells are spread throughout the medium. Visually check the detached cells and afterwards keep the flask on the lid of a round petri dish to keep it flat.
4. Finally, place the flask inside the incubator and leave it undisturbed in a 5% CO₂ incubator at 37 °C for 7 days.
5. Following a week of incubation, invert the flask and check for DFAT cells. Next, assess DFAT cell generation proficiency as described¹⁰ since at this point fibroblast-like cells (DFAT cells) are located in regions distinct from fat cells.
6. Add 5 ml of DCM or DMEM supplemented with 20% FBS to the flask following removal of the previous cell culture medium. Allow the DFAT cells to grow for another week after changing the medium.
7. Using a cell counter, count the number of DFAT cells generated under various cell culture conditions (DCM vs. DMEM).

Results

In this study, the method and toolkit for DFAT cell generation was improved (Figure 1). Our method allows us to generate DFAT cells using both DCM and DMEM medium containing 20% FBS (Figure 2A). As such, we compared the efficiency of DCM and DMEM in generating DFAT cells. In this regard, DCM enhanced DFAT cell proliferation by three times when compared to DMEM, regardless of the number of adipocytes (Figure 2A and B). Usi...

Discussion

Mature adipocytes that undergo in vitro dedifferentiation, a process known as ceiling culture, may revert to a more primitive phenotype and gain proliferative abilities. These cells are referred to as dedifferentiated fat (DFAT) cells. The multilineage differentiation potential of DFAT cells was evaluated. Flow cytometry analysis and gene expression analysis revealed that DFAT cells were highly homogeneous in comparison to ASCs⁶. In fact, the cell-surface antigen profile of DFAT cells is very similar ...

Materials

Name	Company	Catalog Number	Comments
CSTI303-MSC medium	CSTI	87-671	This medium is defined as DCM in the text
PBS(-)	Wako	166-23555	It does not contain Mg2+ and Ca2+
DMEM medium	Gibco	11965-092
Fetal Bovine Serum	Sigma	172012
Collagenase type II	Sigma	C-6885
Scissors	Takasago Medical Industry Co., Ltd	TKZ-F2194-1
Shaker	TAITEC	Bioshaker V.BR-36
Falcon Cell Strainer 100 μm Yellow	CORNING LIFE SCIENCES	DL 352360
Falcon 12.5 cm² Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap	CORNING LIFE SCIENCES	353107
18 G needle	NIPRO	02-002
20 ml Syringe	NIPRO	08-753
Z Series Coulter Counter	BECKMAN COULTER	383550