Adipose tissue derived stem cells: isolation, in vitro culture and potential uses in dermatology

Received on: 10/12/2010
Approved on: 10/01/2011

This study was carried out at the
Dermatology Department of the Hospital do
Servidor Público Municipal de São Paulo in
conjunction with the Molecular
Morphophysiology and Development
Laboratory of the Basic Sciences Department
of the Faculdade de Zootecnia e Engenharia
de Alimentos, Universidade de São Paulo
(USP) – São Paulo (SP), Brazil.

Conflicts of interest: none
Financial support: none

Abstract

There are extensive reports on the characteristics and therapeutic potential of adult mesenchymal stem cells. The possibility of isolating them from excess adipose tissue obtained from other procedures is promising and extremely desirable. However, the protocols for such applications are still described in a summarized, rather than standardized, manner.Thus, the objective of this paper is to present a simple, practical and effective protocol for the collection of adipose tissue in the hospital environment, and for the isolation and in vitro cultivation of adult mesenchymal stem cells obtained from adipose tissue in a laboratory, with the purpose of discussing the potential uses of this technique in the field of dermatology.

Keywords: ADULT STEM CELLS, DERMATOLOGY, STEM CELLS, ADIPOSE TISSUE, TISSUE THERAPY

INTRODUCTION

The scientific innovation and therapeutic potential of stem cells derived from adult tissue for the scientific and medical fields has been discussed for several years.These adult stem cells (ASCs) appear to play a role in the tissues’ natural renewal 1 2.This therapeutic potential is based on intrinsic properties such as clonogenicity (the ability to detect low density cells and activate their duplication capacity), multipotentiality (the potential to originate a great number of more specialized cells), and autorenovation (the capacity to generate new multipotent cells). 3-7 Since these properties were highlighted and summarized in a Science magazine editorial in 1999 8, the scientific community’s interest in studying these cells has steadily grown, due to the enormous potential of application in tissue engineering and genetic therapy 9.

Studies related to cellular therapy are being increasingly published, revealing potential therapies for regenerative medicine – which focuses on the treatment of lesions and disorders – aimed at restoring the functions of damaged or lost cells and tissues through substitution or protection 10. Currently, cellular therapy with adult mesenchymal cells is understood to offer advantages over other methods in tissue repair, resulting in high quality regeneration without the formation of scars or fibrosis. In addition, since it is an autologous transplant it presents a lower risk of rejection and transmission of disorders than other possible exogenous sources of stem cells, such as embryonal cells. 3,4,10-15

Studies carried out by Meirelles and Nardi 16, Haynesworth and others 17, Majumdar and others 18, Romanov and colleagues, 19 and Campagnoli et al. 20 demonstrated that mesenchymal stem cells (MSCs) isolated from bone marrow are capable of self-renovation and differentiation into a variety of conjunctive, osseous, adipose and muscular tissues lineages, allowing their operational classification. Meirelles and colleagues5 showed that cells with MSC characteristics can be derived and cultured in vitro from different organs and tissues such as the brain, spleen, liver, kidneys, lungs, bone marrow, muscles, thymus and pancreas. The presence of stem cells in human adipose tissue had already been reported by Zuk and others 21, who compared the differentiation potential of these cells with MSCs isolated from bone marrow and proved they are similar 22.

It has already been demonstrated that a stem cell population derived from adipose tissue digested with collagenase – also called stromal vascular fraction – is capable of differentiating into several cellular lineages, including adipose tissue, cartilage, bone, skeletal musculature, neuronal cells, endothelial cells, cardiomyocytes and smooth muscle tissue cells 3, 19, 20, 23-27. Because it is easy to obtain stem cells from adipose tissue with minimal patient discomfort – and many times with greater proliferative capacity than those derived from bone marrow cells – adipose tissue can be considered an ideal source of autologous stem cells.The greater proliferative capacity can be intrinsic to the cells or result from a greater density of stem cells in the initial population. Thus, the less invasive, less painful collection process, combined with the possibility of retrieving a considerable amount of mesenchymal cells (enough to avoid extensive in vitro expansion), generate an effective clinical potential for cells derived from adipose tissue, compared to other methodologies.

In this way, adipose tissue has become a considerably attractive source of MSCs in surgical and regenerative medicine for cellular therapy procedures – especially in surgical and cosmetic dermatology – because it can be obtained in great amounts from a material that would otherwise be discarded in liposuction surgeries 28.

This article’s objective is to describe the procedures for obtaining material that has been aspirated through surgical intervention, and isolating and cultivating mesenchymal cells.

COLLECTION, ISOLATION AND CULTIVATION: SUGGESTED PROTOCOL

Adipose tissue collection

The discarded fat from liposuction procedures is used to collect MSCs. The adipose tissue collection procedure begins with a careful study of the site to be targeted. Photographic documentation and marking of donor areas must then be carried out. According to Jurgens and colleagues 29 and Almeida and others 30, the donor areas that offer greater efficiency in terms of stromal vascular fraction cells are located in the trunk, followed by the limbs. In practice, however, the most frequent donor areas are the outer thigh, hips and abdomen regions.

In preparation for the lipoaspirate collection, the asepsis and antisepsis of the donor region is carried out, followed by the application of tumescent anesthesia (180 ml 0.9% saline solution, 10 ml 2% lidocaine hydrochloride without vasoconstrictor, 10 ml 8.4% sodium bicarbonate solution and 0.4 ml Epinephrine diluted to 1/1,000) 31, 32.

After accomplishment of a small anesthetic area in the donor area with 2% lidocaine, an incision with blade 11 is carried out and a 3 mm liposuction cannula with three orifices in the extremity, connected to 10 ml syringes, is inserted.

Around 30 ml of fat are manually aspirated under low pressure (atraumatic). If necessary, the closure is carried out with a suture (simple stitch with 6-0 mono nylon thread) at the site of introduction of the cannula, after massaging the area in order to drain fluids and blood.

The syringes containing the material collected are cleansed two or three times with sterile saline solution, sealed and left to decant for 30 minutes. Afterwards, the supernatant liquid is discarded, and the precipitate is obtained (Figure 1).The syringes containing the remaining fat are sealed and sent to the laboratory for processing.

Digestion of the adipose tissue, isolation and cultivation of mesenchymal cells

Several authors have described different techniques for this phase of the procedure 24,26,33. Rodbell and others 33 introduced the initial method to isolate cells from adipose tissue in laboratory animals. The protocol was later adapted and used with success in human adipose tissue samples 24,26.

The most commonly used methodology for isolating MSCs from lipoaspirates was first published by Zuk and colleagues24, who describe adipose tissue aspiration, digestion, cultivation and characterization of mesenchymal cells processes. The method described below is based on the Zuk technique.

Reagents used
- Phosphate buffered saline (PBS)
- Collagenase IV (SIGMA® Sao Paulo, Brasil)
- Cell culture medium DMEM
- Characterized fetal bovine serum (FBS, Hyclone®)
- Amikacin sulphate (Klebicil, Greenpharma®)
- Amphotericin B (Invitrogen®)
- Substitute of trypsin TrypLE™ express (Invitrogen®)
- Dimethyl sulphoxide (DMSO, Sigma®)

Instruments and equipment used
- 60 mm cell culture dish (TPP®)
- 15 ml and 50 ml conical tubes (TPP®)
- Pipettes (Eppendorf®)
- Pipette tips (TPP®)
- Centrifuge (Baby I centrifuge mod. 206, Fanem®)
- Laminar flow chamber (Trox technik®)
- Incubator (Forma Scientific®)
- Inverted microscope (Leica DM IRB®)
- Mr. Frosty (Nalgene®)
- Neubauer Chamber (Boeco®, Germany)

Procedure

All laboratory procedures are carried out in a laminar flow chamber under sterile conditions (Figure 2).The lipoaspirated adipose tissue should be successively cleansed in PBS solution so that MSCs can be obtained. After the cleansing process, the material undergoes an enzymatic digestion process with collagenase (0.010 g/ml of collagenase IV) in culture medium DMEM, supplemented with 83.4 µ g/ml of amikacin sulphate and 1% amphotericin B.The collagenase solution with the adipose tissue must then be kept in an incubator at 37ºC, with 5% CO2 for 3 hours, under controlled humidity.

After the enzymatic incubation period, the resulting solution is cleansed in culture medium supplemented with 10% FBS and antibiotic, centrifuged at 300 g for 5 minutes, and the supernatant fluid is discarded (adipose fraction). The resulting precipitate is then re-suspended in the culture medium mentioned above, and is subsequently seeded in 60 mm dishes and kept in an incubator at controlled temperature (37ºC) and CO2 atmosphere (5% in air).

After the first 48 hours, the culture medium is changed every other day, with the changes taking place until the cells reach a 70% confluence, then being transfered to plates. These procedures include the cellular trypsinization – the incubation of the cultivation dish with the trypsin enzyme substitute for about 10 minutes, and the inactivation of the process with half DMEM, supplemented with FBS. At this point the cells are transferred to the 15 ml capacity centrifuge’s conical tubes and centrifuged at 300 g for 8 minutes; the supernatant fluid is discarded, the precipitate re-suspended, the concentration is estimated through the Neubauer Chamber, and the cells transferred again to plates. The in vitro culture must be followed up and documented with photographs taken using an inverted microscope (Figure 3).

The preservation and maintenance of the cellular culture is achieved through the freezing process, carried out with the trypsinization stage, as previously described: the supernatant fluid is removed while the resulting precipitate is re-suspended in a previously prepared medium for freezing (DMEM supplemented with 10% of FBS, 10% of DMSO and antibiotic) in a concentration of approximately 1 x 106 cells/ml, distributed in cryotubes. The cryotubes are then allocated in devices specific for freezing (Mr. Frosty) which allow a freezing curve of -1ºC per minute when subjected to a temperature of -80ºC. The device containing the cryotubes is kept in that state for 4 to 24 hours, and is then transferred to a liquid nitrogen tank, where it remains cryopreserved and stored indefinitely.

PERSPECTIVES/OUTLOOK

Cutaneous aging is a challenge for medicine. Plastic surgeons and dermatologists are constantly required to solve problems such as filling wrinkles and deep creases.Therefore several studies are being conducted on mesenchymal cells originally from aspirated adipose tissue. Not only are they easier to obtain than other cells, but there are good regeneration outcomes in the site where these cells were applied under cellular therapy protocols 6. Additionally, some clinical trials demonstrate that these cells differentiate into cells of the resident tissue 34, even though it is currently believed that a possible trophic factor for these cells is the increased tissular regeneration that occurs after injuries 35.

According to Shiffman and Mirrafati 36, the success of the procedure depends directly on the collection, clean-up and material re-injection techniques. However, a protocol defining the best way of processing fat for a long-term graft, independently of the injected area, has not yet been standardized 37.

The combination of MSCs and fat grafts aimed at increasing breast size has recently been shown to promote satisfactory results, without serious complications 38, for it offers the possibility of a long-term filling 39; the adipocytes’ survival is one of the main factors directly interfering in the success of the grafts 24, 27 37.

According to Radovan Borojevic 41, MSC-based cellular therapies are increasingly used in reparative surgeries, especially to repair aesthetic defects such as acne scars, rhytids, and cutaneous aging, among others. It is important to highlight that therapies that use stem cells for rejuvenation do not prevent the natural aging process. Nevertheless, cellular therapy could make it more gradual, resulting in a better quality of life regarding the human body’s functionality and aesthetics.

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