Qualitative and quantitative analysis of rabbit ’ s fat mesenchymal stem cells

Purpose: To present an experimental model of qualitative and quantitative analysis of mesenchymal stem cells from fat of rabbits obtained by lipectomy . The fat could be a great source for obtaining mesenchymal stem cells and to create conditions for repairing injured tissues by bioengineering. Methods: New Zealand rabbits (n= 10) adipose panicle (2-3 cm) were removed by lipectomy , frag ented and washed with PBS and enzymatically dissociated with trypsin/EDT A. Lately, these cells were incubated in culture medium DMEM and after 20 days, was performed quantitative analysis of the accession of first and second mesenchymal cells in cell culture bottles. Results: The fat total cells (CTF) were 1.62 x10 6 cells/mL and presented 98% of viability . These cells were taken for cultivation and after 20 days were counted 2.88 x10 6 cells/mL MSC. The same was done and after 20 days we quantified 4.28 x10 6 cells/mL MSC. Conclusion: The lipectomy of adipose panicule is a very satisfactory method to extract stem cells from fat, quantitatively and qualitatively .


Introduction
Scientific and medical experts have the hope to promoting the legendary concept of regenerative medicine to develop therapies restoring injured tissues and acting on the nervous system lesions central 1,2 .This hope becomes more real each day with the use of advanced technologies such as cell culture, cell therapy, gene therapy and bioengineering, and the adult stem cells (ASC) source of these therapies.
The ASC defined as cells "wild" have the ability to selfrenew and to differentiate into specialized cells 3 (Figure 1).These cells can be found in various niches such as bone marrow, peripheral blood, umbilical cord blood and placental (SCUP), dental pulp, hair follicle and fat, the latter objective of this study.The ASC are divided into two groups: mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC).
MSC found in the bone marrow, also called stromal stem cells, represent 0.01% (10 -2 ) to 0.001% (10 -3 ) of nucleated bone marrow cells.These cells have great capacity for renewal and can differentiate into connective tissue cells such as osteoblasts, condroblasts, adipocytes, fibroblasts and myoblasts 3,4 .
The HSC are derived from mesoderm 5 .The hematopoietic progenitors accumulate around the blood islands initially, migrating to the fetal liver reaching the bone marrow 6,7 .
In the past 20 years, therapy with ASC of umbilical cord has been used successfully in patients with deficiency in bone marrow.The umbilical cord blood offer advantages over the bone marrow, it does not require a perfect compatibility with human leukocyte antigen (HLA).Moreover, this tissue shows a lower incidence of rejection, can be used allogeneic 8 .However, to obtain ASC of umbilical cord becomes limited, as can be gained only after the birth of the individual.
In this sense it is necessary to obtain ASC alternative sources, such as the adipose tissue presented in this study [9][10][11][12] .Therefore, the objective of this study was to perform a qualitative and quantitative analysis of mesenchymal stem cells extracted from fat tissue of rabbits by lipectomy.

Methods
This study was approved by the Research Ethics Committee of the Federal University of Sao Paulo (UNIFESP).New Zealand rabbits were used (n=10), 3 months, weighing approximately 3.5 kg from the animal colony of the Institute of Applied Sciences in Otorhinolaryngology (ICAO), Sao Paulo.

Collection of adipose tissue
The animals were submitted to general anesthesia with ketamine and zoletil (0.4 ml/kg each), by intramuscular injection.Subsequently, a longitudinal incision was performed 2 to 3 cm in the dorso-medial line, where is located the adipose panicle, in which the removed fat desired (lipectomy) (Figure 2).

Separation, cultivation and quantification of ASCs
After collection, the adipose tissue was fragmented (explant technique) into small pieces (about 1 cm) and washed with PBS supplemented with penicillin and streptomycin (1%) (Figure 3).Subsequently, the fragments were enzymatically dissociated with trypsin / EDTA.After the enzymatic process the material was subjected to centrifugation to the rupture of adipocytes to obtain a button of blood cells from the total fat (CTF) which were counted in a Neubauer chamber.Then were the qualitative part of the study through the cell viability using the dye trypan blue, which is associated to DNA when cell membrane rupture.After confirmation of cell viability, these cells were incubated in culture medium DMEM (high glucose, supplemented with fetal bovine serum and 10%) at 37°C with 5% CO ² .After 20 days, was performed quantitative analysis of the accession of mesenchymal cells (MSC 1pd) in cell culture bottles.
By the enzymatic method (trypsin + EDTA) performed the resuspension of MSC to wrap them in two bottles of the second culture passage (MSC 2pd), and again after 20 days quantify them in a Neubauer chamber.

Quantitative analysis of blood cells of total fat (CTF) before and after culture MSCs
After extraction of CTF, the counting was done in Neubauer chamber which were 1.62 ± 0.17 (x10 6 cel/mL) CTF.These cells were taken for culture and after 20 days were counted 2.88 ± 0.24 (x10 6 cel/mL) MSC (1 pd).These cells were resuspended with trypsin and placed in two bottles which were kept in culture for another 20 days and counted again, quantified 4.28 ± 0.36 (x10 6 cel/mL) MSC (2 pd), as shown in Figure 4.

Cell viability
The CTF (Figure 5) and MSC (data not show) cell viability was done by the reaction with Trypan Blue showing a viability of 98%.

Discussion
This study showed that the enzymatic treatment of rabbit adipose panicle with Trypsin / EDTA resulted in obtaining a large number of blood cells of total fat (CTF).Unfortunately these cells can not be characterized as mesenchymal and hematopoietic cells by using optical microscopy.Because of the population of cells extracted of adipose panicule observed in microscope show appearance of mononuclear and granular cells, we called them by blood cells from total fat (CTF).This work has shown that we can extract 1.62 x10 6 cells/mL of CTF from adipose panicule with good viability.In the same way Forriol and Esparza 14 suggest that fat we can extract 4x10 7 cell/mL of CTF.
Despite we have obtained a good number of CTF, we can also amplify these cells using a culture method.These cells, when maintained in culture in an appropriate medium, were amplified by 77% (on relation of CTF) showing a good option to apply these cells to repair injuries.Because of a few studies of fat stem cells, we can not compare exactly the proliferation of these cells types.However, other authors have amplified by about 40% the blood cells extracted from bone marrow when these cells were maintained on culture [14][15][16] .
The cells on culture were also show to be able to adhere on culture bottle, extending and converging like a tissue, forming a fibroblastoid colony (CFU-F), like other authors have observed 13- 16 .
Is important to note that the second passage also increase the quantity of MSC by 164% (on relation of CTF) showing that these cells have a good capacity of proliferation, like other authors have observed [20][21][22][23][24] .These authors have also observed that these cells maintain the same physiology until five passages 23,24 .

Conclusion
The fat can be an excellent alternative way to obtain stem cells with good viability.These cells have greatly increased its population even forming a colony.The fact of forming a colony suggests that these cells have the capacity to form tissues and organs.

FIGURE 1 -FIGURE 2 -FIGURE 3 -
FIGURE 1 -The pluripotential ASC.(Modified from Leri et al. 13 ) FIGURE 2 -Figures representing.A. dorsal region of rabbit; B. incision of rabbit dorsal region exposing the fat; C. collecting fat from dorsal region; D. Fat on tube with solution of phosphatebuffered saline (PBS)

FIGURE 4 -
FIGURE 4 -Histogram representing the total cells extracted from adipose panicle rabbit (CTF) and cultivated mesenchymal stem cells after first passage (MSC -1pd) and second passage (MSC -2pd).The columns and bars respectively indicate mean ± standard error of mean of cells from 10 rabbits.Statistically different from the CTF, * 0.001 and ** p ≤ p ≤ 0.0001 (Student t-test).

FIGURE 5 -
FIGURE 5 -Figures represent the cell viability of CTF with trypan Blue.The arrows represent CTF who are colorless.