|Year : 2016 | Volume
| Issue : 1 | Page : 116-121
Adipose-derived stem cells: An optimized protocol for isolation and proliferation
Mahdieh Ghiasi, Reza Tabatabaei Qomi, Naser Kalhor, Mohsen Sheykhhasan
Stem cell laboratory, Stem cell laboratory, The Academic Center for Education, Culture and Research (ACECR)- Qom Branch, shabnam avenue, isar square, Qom, Iran
|Date of Web Publication||5-Jul-2017|
Stem cell laboratory, The Academic Center for Education, Culture and Research (ACECR)- Qom Branch, shabnam avenue, isar square, Qom
Source of Support: None, Conflict of Interest: None
Background and Aims: Current advances in the researches on the stem cells has opened new approaches for their apply in tissue engineering and clinical trials. The most common sources of stem cells are adult and embryonic stem cells. Due to ethical issues, embryonic stem cells use in research has been hotly debated. Unlike embryonic stem cells, adult stem cell have not ethical problem for clinical and research. The purpose of present study was to stem cells isolation and proliferation from the human dipose tissue.
Materials and Methods: In this study, stem cells were successfully isolated from human adipose tissue by digestion with type I collagenase enzymes. In summary, adipose tissue were digested by type I collagenase enzyme. Subsequently, the cell solution was centrifuged for mature adipocytes and debris elimination and obtained sedimentation was cultured in culture medium contains Dulbecco's Modified Eagle's Medium (DMEM) via 1% penicillin/streptomycin antibiotics and 10% fetal bovine serum (FBS) at 37°C, 5% CO2 and 95% humidity.
Results: in this study, ADSCs were successfully isolated and proliferated. Human ADSCs were able to divide in our culture mediums.
Conclusion: The results of the current study were suggesting that this adipose-derived stem cell isolation protocol provides an effective and improved method for isolation and proliferation of these cells in order to tissue engineering application.
Keywords: Adipose-derived stem cells, Optimized protocol, Cell isolation, Cell proliferation
|How to cite this article:|
Ghiasi M, Qomi RT, Kalhor N, Sheykhhasan M. Adipose-derived stem cells: An optimized protocol for isolation and proliferation. Acta Med Int 2016;3:116-21
| Introduction|| |
Recent studies have shown that the one of the most regenerative system in the body is indicated by stem cells. These cells are available in different part of the body, such as blood, adipose and etc., These cells have attractive properties such as multilineage differentiation and self- renewal capability. Adult stem cells, such as adipose- derived stem cells (ADSCs) are one of the most common stem cells because these cells have not ethically challenge.
ADSCs are usually isolated by enzymatic digestion of different tissue such as abdominal subcutaneous adipose., Mesenchymal stem cells are well-known by self-renewal capability, to differentiate into different cell types, to adhere to flask bottom and to expression of variety surface markers., MSCs observed and isolated from all post-natal tissue and organ. Recently, the development of stem cell technology has provided significant hope to treat or prevent a disease and tissue damages and finally supplied huge progress in regenerative medicine and tissue engineering.,,
Stem cell technology is an advanced approach to the therapy of diseases and damages that uses suitable cell sources. Studies using this method have been suitable alternative to conventional clinical method. Stem cells are cells with the capability to divide and make a lot of identical stem cells or to specialize and type specific cells of bodily tissues. Recently, the development of stem cell technology has provided significant hope for tissue engineering and regenerative medicine. Try to optimize of isolation and expansion adult stem cells protocols is a critical step in the expansion of regenerative medicine methods for tissue repair or regeneration for the therapy of diseases and damages.,,,,,,,,,,,,,, Adipose-derived stem cells supply a plentiful and suitable source of adult stem cells for regenerative medicine and tissue engineering application.,,, Several methods have been used during the years to treat a variety of body damages and lesions.,, Among them, recently, the emerging progress in the field of stem cell technology is to treat the diseases and lesions by using different cell sources, which provide an appropriate support for repair of lesions and stimuli for tissue regeneration. This will ensure the availability of autologous ADSCs for research, trial and clinical applications in the future. ADSCs have an appropriate cell source for regenerative medicine.,
In this study, adipose-derived stem cells were successfully isolated by enzymes digestion.
The purpose of this in vitro study was to establish an improved protocol for ADSC isolation and proliferation. We also compared the properties of different protocols in isolation and proliferation of adipose-derived stem cells.
| Materials and Methods|| |
Human abdominal subcutaneous adipose sample was obtained from 3 patients undergoing of elective surgery after obtaining written consent from patients [Figure 1]. Cell isolation was performed from the adipose tissue samples. Initially, the adipose tissue sample was mechanically segmented and connective tissue and blood vessel were removed carefully from this sample. The obtained pieces were rinsed with phosphate buffered saline (PBS) several times and were digested with type I collagenase (1.5 mg/1g adipose tissue) at 37°C for 45-60 min. Then, the cell suspension was centrifuged at 1800 rpm for 10 min and the cell pellet was cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with penicillin streptomycin and 10% FBS, and incubated at 37°C with 5% CO2 and 95% humidity. The medium was changed at least twice per week until the cells reach up to 80% confluence. In this method, A large number (approximately 200,000) of adipose-derived stem cells (ADSCs) were obtained per gram of adipose tissue [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d,[Figure 2]e,[Figure 2]f,[Figure 2]g,[Figure 2]h,[Figure 2]i,[Figure 2]j. Cells were passaged at 80% confluence by washing twice with PBS and were incubated in trypsin/EDTA at 37°C for 3 min. After the cells reached confluence at the third passage, ADSCs were frozen according to current study protocol. Cold freezing medium contains about 50% fetal bovine serum, 10% dimethyl sulfoxide (DMSO) and 40% DMEM. The viable cells were preserved in a final concentration of up to 1× 106 cells/ml at -196°C in liquid nitrogen tank for a long time.
|Figure 1: Schematic image demonstrating of the human abdominal subcutaneous adipose tissue and it's components|
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|Figure 2: different steps of adipose-derived stem cells isolation (a-j). This schematic describes the isolation process of adipose-derived stem cells from harvested whole human adipose tissue|
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| Results|| |
In the current study, we succeeded in isolating and proliferating stem cells from the subcutaneous adipose tissue using an easy and effective protocol. In this work, cell adhesion observed 2-3 day after initiation of the primary culture. Also, isolated cells having a spindle-shape and gradually they become elongated. At last, these cells showed a typical fibroblast morphology (fibroblast-like shape) under inverted phase-contrast microscope. The cells were started to adhesion and proliferation. Two weeks after cell isolation, ADSCs reached to up to 80% confluent rate. The cells had a higher proliferation rate in the next passages to passage three and obtained at about 90% confluence after the seven days [Figure 3]. These cells had a rapid expansion in vitro condition. Also, ADSCs displayed positive staining for the mesenchymal surface markers CD44, CD90, CD105 [Figure 4]. ADSCs at passage four that showed high-level of CD44, CD90, CD105 expression. More than 70% of ADSCs expressed the mesenchymal-specific markers (CD44, CD90, CD105). In contrast, only a small proportion (less than 5%) of ADSCs expressed the hematopoietic marker CD34 [Figure 5] & [Table 1].
|Figure 3: Human adipose-derived stem cells at different passages. (a) Passage 0, (b) Passage 1, (c) Passage 2 and (d) Passage 3|
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|Figure 4: Schematic image of ADSCs specific surface markers (CD44, CD90, CD105)|
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|Figure 5: Expression of ADS cells specific markers (CD44, CD90, CD105) on primary culture of ADSCs by flow cytometry method. CD34 antibody was used as negative marker|
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|Table 1: Flow cytometry analysis of adipose-derived stem cells (ADSCs) at passage 4|
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ADSCs were successfully stored more than six months in liquid nitrogen tank [Figure 6].
|Figure 6: Schematic image demonstration of successful storage of ADSCs in liquid nitrogen tank|
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| Disscusion|| |
In this study, we showed the potential of this protocol for adipose-derived stem cells isolation. In the present study, in primary isolated cells, more than 70% of the cell population positively expressed mesenchymal surface markers (CD 44, CD90, and CD105). Also, these cell populations negatively expressed hematopoetic marker (CD34). The characterization of isolated cells was confirmed as ADSC by flow cytometry method. These results were supported by many studies result that reported previously.,,,, Our current method was developed by using of a simple protocol of washing adipose tissue. Also, in this method the processing time was modified in comparison with the enzymatic methods. In this work, we succeeded in isolating and proliferating these cells in the try to use them in tissue engineering application in the future.
The stem cells of the adipose tissue can represent a critical source of cells with multipotential properties.,, The optimization of the isolation and culture conditions may increase the comparability of the results from different research laboratories. Although, the essential elements in optimal isolation of stem cells are the type of used enzymes and or materials and duration of different steps and the centrifugation process, but no unique and optimized isolation protocol for ADSCs has been accepted overall.
The primary techniques to isolate ADSCs were pioneered by rodbell et al utilizing rat adipose tissue. This classic method to isolate human adipocyte progenitors was modified by Van, Roncari, Deslex, Hauner and others.,,,
Different methods were assessed for ADSCs isolation.,,,,,,,,,,,,,,,,,,,,,,,, Most Common methods for ADSCs isolation is consisted of washing the adipose tissue, digestion with collagenase and subsequently centrifugation process steps.
The first important step in isolation adipose-derived stem cells is washing process that this process can be affected on ADSCs's heterogeneity.
Different ADSCs isolation protocols were used in variance components such as Ca2+- and Mg2+-free Hank's balanced salt solution containing sodium HEPES, Phosphate Saline Serum (PBS) and Normal Saline Serum in washing step., In the current study, we used to Phosphate Saline Serum (PBS) for washing the adipose tissue sample.
Katz et al were developed a novel protocol for adipose-derived stem cells isolation with modifying in collagenase digestion and tissue washing steps that were largely corroborated our present study.
Such as our protocol, The recent protocols for human adipose-derived stem cell isolation have been used a collagenase digestion followed by centrifugation process to separate the stromal vascular fraction (SVF) from pre-adipocytes.
In some of the protocols, collagenase enzyme such as 0.075% collagenase type I, 0.3% collagenase type I and 0.6% collagenase type I was replaced with trypsin.,, Some protocols such developed protocol by shah et al was used a non-enzymatic method.,,,
For example, in a study, a mechanical strategy was used for isolating adipose-derived stem cells from a conventional liposuction method by raposio et al. In this study, no enzymes such as collagenase and animal-derived materials were used. Also, Developed protocol by baptista et al was reply on mechanical shaking incubation plus to red blood cell lysis solution, simultaneously.
ADSCs proliferation can be highly influenced by basal medium, glucose concentration, Quality of FBS, cell plating, and cell density. Cheng et al were used the KNAC medium culture for growth of human ADSCs with a lower calcium rate. This experiment that use of K-NAC medium for cell growth have been significantly increased in cell proliferation ability.
| Conclusion|| |
In conclusion, our findings propose that this protocol can be utilized for the isolation of ADSCs, particularly when purpose of study related to tissue engineering strategies. Also, this protocol can be easier, cheaper, and faster method than other current procedures.
| Acknowledgements|| |
We thank The Academic Center for Education, Culture and Research (ACECR) - Qom Branch as the financial and executive sponsor.
| References|| |
Tabatabaei-Qomi R, Sheykh-Hasan M, Fazaely H, Kalhor N, Ghiasi M. Development of a PCR assay to detect mycoplasma contamination in cord blood hematopoietic stem cells. Iranian journal of microbiology. 2014; 6(4):281–284.
Araña M, Mazo M, Aranda P, Pelacho B, Prosper F. Adipose tissue-derived mesenchymal stem cells: isolation, expansion, and characterization. Methods Mol Biol. 2013;1036:47–61. doi: 10.1007/978-1-62703-511-8_4.
Baptista LS, do Amaral RJ, Carias RB, Aniceto M, Claudio-da-Silva C, Borojevic R. An alternative method for the isolation of mesenchymal stromal cells derived from lipoaspirate samples.Cytotherapy. 2009;11(6):706–15. doi: 10.3109/14653240902981144.
Buehrer BM, Cheatham B. Isolation and characterization of human adipose-derived stem cells for use in tissue engineering. Methods Mol Biol. 2013;1001:1–11. doi: 10.1007/978-1-62703-363-3_1.
Sheykhhasan M, Tabatabaei Qomi SR, Kalhor N, Ghiasi M. Isolation and maintenance of canine Adipose-derived Mesenchymal Stem Cells in order to tissue engineering application. International Journal of Advanced Biological Science and Engineering. 2014;1(2):86–97.
Ghiasi M, Fazaely H, Asaii E, Sheykhhasan M. In Vitro Maturation of Human Oocytes using Conditioned Medium of Mesenchymal Stem Cells and Formation of Embryo by Use of ICSI. SMU Medical Journal. 2014; 1(1):89–98.
Sheykh Hasan M, Kalhor N, Tabatabaei Qomi SR, Ghiasi M. Optimization method for isolation and culture of chondrocytes in human nasal cartilage tissue. International Journal of Advanced Biological Science and Engineering. 2014;1(2):74–85.
Ghiasi M, Tabatabaei Qomi R, Nikbakht M, Sheykhhasan M. Expression of collagen type I and II, aggrecan and SOX9 genes in mesenchymal stem cells on different bioscaffolds. Tehran University Medical Journal. 2015; 73(3): 158–167.
Tabatabaei Qomi R, Sheykhhasan M, Kalhor N, Ghiasi M. Chondrogenic Differentiation of Human Adipose-derived Mesenchymal Stem Cells Using Fibrin Hydrogel Scaffold. J Mazandaran Univ Med Sci. 2015; 25(123): 21–30
Cheng K-H, Kuo T-L, Kuo K-K, Hsiao C-C. Human adipose-derived stem cells: Isolation, characterization and current application in regeneration medicine. Genomic Medicine, Biomarkers, and Health Sciences. 2011;3(2):53–62.
Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adipose-derived stem cells: isolation, expansion and differentiation. Methods. 2008 Jun;45(2):115–20. doi: 10.1016/j.ymeth.2008.03.006. Epub 2008 May 29.
Locke M, Windsor J, Dunbar PR. Human adipose-derived stem cells: isolation, characterization and applications in surgery. J Surg.2009; 79:235–244.
Mailey B, Hosseini A, Baker J, Young A, Alfonso Z, Hicok K, Wallace AM, Cohen SR. Adipose-derived stem cells: methods for isolation and applications for clinical use. Methods Mol Biol. 2014;1210:161–81. doi: 10.1007/978-1-4939-1435-7_13.
Raposio E, Caruana G, Bonomini S, Libondi G. A novel and effective strategy for the isolation of adipose-derived stem cells: minimally manipulated adipose-derived stem cells for more rapid and safe stem cell therapy. Plast Reconstr Surg. 2014 Jun;133(6):1406–9. doi: 10.1097/PRS.0000000000000170.
Doi K, Kuno S, Kobayashi A, Hamabuchi T, Kato H, Kinoshita K, Eto H, Aoi N, Yoshimura K. Enrichment isolation of adipose-derived stem/stromal cells from the liquid portion of liposuction aspirates with the use of an adherent column. Cytotherapy. 2014 Mar;16(3):381–91. doi: 10.1016/j.jcyt.2013.09.002. Epub 2013 Nov 12.
Dubois SG, Floyd EZ, Zvonic S, Kilroy G, Wu X, Carling S, Halvorsen YD, Ravussin E, Gimble JM. Isolation of human adipose-derived stem cells from biopsies and liposuction specimens. Methods Mol Biol. 2008;449:69–79. doi: 10.1007/978-1- 60327-169-1_5.
Estes BT, Diekman BO, Gimble JM, Guilak F. Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc. 2010 Jul;5(7):1294–311. doi: 10.1038/ nprot.2010.81. Epub 2010 Jun 17.
Francis MP, Sachs PC, Elmore LW, Holt SE. Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction. Organogenesis. 2010 Jan-Mar;6(1):11–4.
Ghorbani A, Jalali SA, Varedi M. Isolation of adipose tissue mesenchymal stem cells without tissue destruction: a non-enzymatic method. Tissue Cell. 2014 Feb;46(1):54–8. doi: 10.1016/j. tice.2013.11.002. Epub 2013 Nov 12.
Gimble J, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy. 2003;5(5):362–9.
Griesche N, Luttmann W, Luttmann A, Stammermann T, Geiger H, Baer PC. A simple modification of the separation method reduces heterogeneity of adipose-derived stem cells. Cells Tissues Organs. 2010;192(2):106–15. doi: 10.1159/000289586. Epub 2010 Feb 24.
Markarian CF, Frey GZ, Silveira MD, Chem EM, Milani AR, Ely PB, Horn AP, Nardi NB, Camassola M. Isolation of adipose-derived stem cells: a comparison among different methods. Biotechnol Lett. 2014 Apr;36(4):693–702. doi: 10.1007/s10529-013-1425-x. Epub 2013 Dec 10.
Odabas S, Elçin AE, Elçin YM. Isolation and characterization of mesenchymal stem cells. Methods Mol Biol. 2014;1109:47–63. doi: 10.1007/978-1-4614-9437-9_3.
Shah FS, Wu X, Dietrich M, Rood J, Gimble JM. A non-enzymatic method for isolating human adipose tissue-derived stromal stem cells. Cytotherapy. 2013 Aug;15(8):979–85. doi: 10.1016/j. jcyt.2013.04.001. Epub 2013 May 29.
Romanov YA, Darevskaya AN, Merzlikina NV, Buravkova LB. Mesenchymal stem cells from human bone marrow and adipose tissue: isolation, characterization, and differentiation potentialities. Bull Exp Biol Med 140(1):138–143, 2005.
Sharifi AM, Ghazanfari R, Tekiyehmaroof N, Sharifi MA. Isolation, Cultivation, Characterization and Expansion of Human Adipose-Derived Mesenchymal Stem Cell for Use in Regenerative Medicine. International Journal of Hematology-Oncology & Stem Cell Researc. 2012; 6(1):1–5.
Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, Aiba-Kojima E. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol. 2006;208:64–76.
Yu G, Floyd ZE, Wu X, Halvorsen YD, Gimble JM. Isolation of human adipose-derived stem cells from lipoaspirates. Methods Mol Biol. 2011;702:17–27. doi: 10.1007/978-1-61737-960-4_2.
Rodbell M. Metabolism of isolated fat cells. Effects of hormone on fat metabolism and lipolysis. J Biol Chem 1964;239:375_380.
Rodriguez AM, Elabd C, Amri EZ, Ailhaud G, Dani C. The human adipose tissue is a source of multipotent stem cells. Biochimie.2005;87(1):125–128.
Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13:4279–4295.
Deslex S, Negrel F, Vannier C et al. Differentiation of human adipocyte precursors in a chemically defined serum-free medium. Int J Obes 1986;10:19–27.
Ghiasi M, Tabatabaei-Qomi R, Kalhor N, Fazaely H, Mehdizadeh Mohammad, sheykh hasan M. The Design of Scaffolds for Use in Tissue Engineering. SMU Medical Journal. 2014; 1(2):261–273.
Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001 Apr;7(2):211–28.
Ghiasi M, Sheykhhasan M, Tabatabaei Qomi R, Kalhor N, Mehdizadeh M. The comparison of natural and synthetic scaffolds protective capabilities as a suitable growth medium for adipose-derived mesenchymal stem cells. New Cellular and Molecular Biotechnology Journal. 2015;5(17): 35–40
Kate AJ, Llull R, Hedrick MH et al. Emerging approaches to the tissue engineering of fat. Clin Plas Surg 1999;26:587–603.
Reshak AH, Shahimin MM, Buang F. Comparative study on human and bovine AT-SC isolation methods. Prog Biophys Mol Biol 2013 Nov;113(2):295–8. doi: 10.1016/j.pbiomolbio.2013.09.001. Epub 2013 Sep 27.
Ghiasi M, Kalhor N, Tabatabaei Qomi R, Sheykhhasan M. The effects of synthetic and natural scaffolds on viability and proliferation of adipose-derived stem cells. FRONTIERS IN LIFE SCIENCE, 2015;8(4):1–12.
Sheykhhasan M, Tabatabaei Qomi R, Ghiasi M. Fibrin Scaffolds Designing in order to Human Adipose-derived Mesenchymal Stem Cells Differentiation to Chondrocytes in the Presence of TGF-ß3. International Journal of Stem Cells 2015;8(2):1–9. http://dx.doi. org/10.15283/ijsc.2015.8.2.1
Sheykhhasan M, Tabatabaei Qomi R, Kalhor N, Mehdizadeh M, Ghiasi M. Evaluation of the ability of natural and synthetic scaffolds in providing an appropriate environment for growth and chondrogenic differentiation of adipose-derived mesenchymal stem cells. indian journal of orthopaedics. 2015;49(5):8–15.
Storck K, Ell J, Regn S, Rittler-Ungetüm B, Mayer H, Schantz T, Müller D, Buchberger M. Optimization of in vitro cultivation strategies for human adipocyte derived stem cells. Adipocyte. 2015:1–7.
Baer PC. Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro. World J Stem Cells. 2014 26;6(3):256–65. doi: 10.4252/wjsc.v6.i3.256.
Van RLR, Bayliss CE, Roncari DAK. Cytological and enzymological characterization of adult human adipocyte precursors in culture. J Clin Invest 1976;58:699–704.
Hauner H, Entenmann G, Wabitsch M et al. Promoting effect of glucocorticoids on the differentiation of human adipocyte precursor cells cultured in a chemically defined medium. J Clin Invest 1989;84:1663–70.
Rada T, Reis RL, Gomes ME. Distinct stem cells subpopulations isolated from human adipose tissue exhibit different chondrogenic and osteogenic differentiation potential. Stem Cell Rev. 2011 Mar;7(1):64–76. doi: 10.1007/s12015-010-9147-0.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]