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Isolation and Cultivation of Rabbit Adipose-derived Stem Cells and the Influence of Platelet-rich Plasma on the Cell Proliferation

Author: ChenJin
Tutor: HuGang
School: Dalian Medical University
Course: Surgery
Keywords: adipose-derived stem cells platelet-rich plasma cell culture cell proliferation
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 7
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Abstract


Background: Autologous fat transplantation has been applied for hundreds of years, and highly concerned by cosmetic and plastic surgeons for the advantages of this technique, such as rich sources of fat cells, easy to get the material, simple operation , good filling shape and without graft rejection, etc. While the definitive evidence of fat survival is still in question (The survival rate is lower than 60%).How to improve the proliferation and differentiation of the fat cells is a key to increase the graft survival rate. Currently there are two significant technical hurdles in the development of adipose tissue engineering. One is looking for the source-rich, easily-obtained, minimum-damaged and proliferation-steadied stem cells;the other is chosen the effective growth factors which are without immune response to the recipient, economic, and with the ability of enhancing the stem cell engraftment.Objective: The purpose of this research is to isolate and culture rabbit adipose-derived stem cells (ADSCs).to observe the biological characteristics of ADSCs in vitro and the potential capabilities of proliferation and differentiation of these cells,and to investigate the impacts of self-derived platelet-rich plasma (PRP) on the ADSCs proliferation.Methods:1. Resect the cervicodorsal adipose tissue from the New Zealand white rabbit. The ADSCs were cultured in vitro using adherent method and collagenase I digestion method. The morphological characteristics of the ADSCs were observed.2. The third passage ADSCs were inducted and differentiated into adipogenic cell line and osteogenic cell line. Oil O red staining and alizarin staining were performed after two and three weeks of induction to identify the differentiation of ADSCs. 3. The rabbit heart blood was collected, and the platelet-rich plasma (PRP) was prepared by two-step-centrifugation method. Platelets in whole blood and PRP were counted by platelet cell count board.4. The third passage of ADSCs was divided into three groups: control group(180μl ADSCs with 20μl DMEM culture media),whole blood group (180μl ADSCs with 20μl plasma)and platelet-rich plasma group(180μl ADSCs with 20μl PRP). The proliferation of ADSCs at three time points (24h, 48h and 72h)were detected by the Cell Counting Kit-8.Results:1. A small amount of elliptical-shaped cells adhered to the plastic surface after 24 hours primary cultivation, and most of the non-adherent cells were erythrocytes and fasciae debris. Remove erythrocytes and cell debris by changing culture media in 48 hours.Some spindle cells adhered to the plastic was observed at that time, and a few of triangle and polygonal shaped cells also scattered in the cell culture. After 3 days,part of adherent cells was elongated, in long spindle shape and gradually increased.Mitosis phenomena were also observed.On the 7th day, 80% -90% cells inosculated and was in circinate-liked growth. Most of the cells were long spindle shape and fibroblast-like in variable size. The proliferation of passage cells was significantly increased compare to primary cells. Cell adherent to the plastic happened within 4-6 hours. The morphology of passage cells was similar to the original generation.2. In two weeks after adipogenic induction, oil red O staining was positive, but negative in control group;in three weeks after osteogenic induction, alizarin red staining was also positive but not in control group.3. The number of the platelet in the whole blood was(313.0±137.5)×10~9/L, while in the PRP was (1267.0±760.2)×10~~9/L, which was 4.08 times of it in the whole blood.4. CCK-8 assay showed that the proliferations of PRP group at 24h、48h、72h were significantly increased comparing to the control group (P<0.01), and also had significant difference (P<0.05) to the whole blood group.Conclusion:1. ADSCs were able to be isolated from rabbit cervicodorsal adipose tissues by collagenase digestion. The ADSCs obtained in our study with rapid and stable cell proliferative ability can be passaged in long-term.2. ADSCs have the potential of multilineage differentiation. Under certain conditions, ADSCs can be induced and differentiated into adipogenic cell and osteoblasts. Therefore, ADSC, as a suitable seed cell can be used in tissue engineering.3. Two-step centrifugation method is an effective method for preparation of PRP. The platelet in rabbit PRP prepared by this method was four times enriched comparing to the platelet in the whole blood.4. Autologous PRP has significantly proliferation-promoting effect on rabbit ADSCs culture in vitro.

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