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Isolation and Expansion of Mesenchymal Stem Cells from Murine Adipose Tissue
从鼠脂肪组织中分离和扩增间充质干细胞   

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Abstract

Mesenchymal stem cells (MSCs) are currently intensively studied due to significant promise which they represent for successful implementations of future cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies of MSC effects in various murine disease models. The appropriate cell preparations with reproducible biological properties are important to minimize variability of results of experimental cell therapies. We describe here a simple protocol for isolation of murine MSCs from adipose tissues and their reproducible multi-log expansion under hypoxia conditions.

Keywords: Mesenchymal stem cells(间充质干细胞), Adipose tissue(脂肪组织), Oxidative stress(氧化应激), Hypoxia(缺氧), Expansion(扩增)

Background

MSCs were identified initially by Friedenstein as cells in bone marrow with fibroblast-like morphology, adherence to the plastic and high self-renewal capacity resulting in formation of fibroblast-like colonies in vitro (Friedenstein et al., 1976; reviewed in Phinney and Sensebé, 2013). The MSCs, due to their potential applications in medicine, are currently one of the most intensively studied adult progenitor cell types. These cells can be isolated from various organs (Murray et al., 2014), and are thought to originate from the blood vessels, either as pericytes or as vessel wall cells. In addition to their capacity to differentiate along osteogenic, adipogenic and chondrogenic lineages, MSCs possess immunomodulating properties and are thought to participate in responses to tissue damage as well as to orchestrate anti-inflammatory reactions through their ability to influence macrophage polarization (Prockop, 2013; Caplan, 2016).

Given these properties, MSCs represent significant promise for successful implementations of future relevant cell therapy clinical protocols. This in turn emphasizes importance of careful preclinical studies with MSCs in various murine disease models. The ability to prepare large numbers of appropriate cell samples with reproducible biological properties is of vital importance for minimization of variability of results during development of MSC-based experimental cell therapies. However, unlike human MSCs that possess strong anti-oxidative defenses and thus grow fairly well under atmospheric oxygen conditions, mouse MSCs are much more sensitive to oxygen stress and have a limited lifespan and expansion capacity when cultured in conventional CO2 incubators. Culturing these cells under hypoxic conditions, on the contrary, significantly extends their lifespan and allows for multi-log expansion, providing sufficient amounts of cell material with reproducible properties for repeated experiments with murine experimental disease models (Boregowda et al., 2012; Krishnappa et al., 2013). In the present paper, we describe a simple protocol for isolation of murine MSCs from adipose tissue, their reproducible expansion under hypoxia conditions, as well as long-term storage.

Materials and Reagents

  1. 6 cm cell culture dish (Greiner Bio One International, catalog number: 628160 )
  2. Sterile pipette filter tips 20 μl (Greiner Bio One International, catalog numbers: 774288 )
  3. Sterile pipette filter tips 200 μl (Greiner Bio One International, catalog numbers: 739288 )
  4. Sterile pipette filter tips 1,000 μl (Greiner Bio One International, catalog numbers: 740288 )
  5. 15 ml centrifuge tube (Greiner Bio One International, catalog number: 188261 )
  6. 50 ml centrifuge tube (Greiner Bio One International, catalog number: 227261 )
  7. 1.8 ml round bottom cryogenic tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 375418 )
  8. 10 cm cell culture dish (Greiner Bio One International, catalog number: 664160 )
  9. 3.5 cm cell culture dish (Greiner Bio One International, catalog number: 627160 )
  10. Cotton wool
  11. Vacuum filter/storage bottle system, 0.22 µm, 500 ml (Corning, catalog number: 431097 )
  12. 0.22 μm syringe filter (Sigma-Aldrich, catalog number: Z741948)
    Manufacturer: GVS, catalog number: 1214220 .
  13. 10 ml syringe (SFM Hospital Products, catalog number: 534235 )
  14. 2 ml serological pipets (Greiner Bio One International, catalog numbers: 710180 )
  15. 5 ml serological pipets (Greiner Bio One International, catalog numbers: 606180 )
  16. 10 ml serological pipets (Greiner Bio One International, catalog numbers: 607107 )
  17. 25 ml serological pipets (Greiner Bio One International, catalog numbers: 760160 )
  18. C57BL/6 mice
  19. Sterile distilled water
  20. Ethanol 96% (Sigma-Aldrich, catalog number: 24105 )
    Note: This product has been discontinued.
  21. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  22. Dimethyl sulfoxice (DMSO) (Sigma-Aldrich, catalog number: D2650 )
  23. Propidium iodide (PI) (Thermo Fisher Scientific, InvitrogenTM, catalog number: P3566 )
  24. Cell culture media components
    1. DMEM low glucose, powder (Thermo Fisher Scientific, GibcoTM, catalog number: 31600083 )
    2. GlutaMax (100x) (Thermo Fisher Scientific, catalog number: 35050038 )
    3. Penicillin-streptomycin (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    4. Sodium hydrogen carbonate cell culture grade (AppliChem, catalog number: A0384 )
  25. Amphotericin B (0.25 mg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  26. Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C2674 )
  27. Fetal bovine serum (GE Healthcare, HyCloneTM, catalog number: SV30160.03 )
  28. Phosphate-buffered saline (PBS), pH 7.4 tablets (Thermo Fisher Scientific, catalog number: 18912014 )
  29. Trypsin from porcine pancreas (Sigma-Aldrich, catalog number: T4799 )
  30. Ethylenediaminetetraacetic acid disodium salt (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  31. DMEM low glucose medium (see Recipes)
  32. MSC isolation medium (see Recipes)
  33. Collagenase solution (see Recipes)
  34. MSC growth medium (see Recipes)
  35. 1x phosphate-buffered saline (PBS) pH 7.4 (see Recipes)
  36. Trypsin solution (see Recipes)

Equipment

  1. Sterilized surgical tools including forceps and scissors
  2. Pipette controller (Corning, catalog number: 4091 )
  3. Automatic single-channel pipettes, 0.5-20, 20-200 and 100-1,000 μl, Gilson-compatible (Gilson)
  4. Analytical balance*
  5. Thermostated shaker (Eppendorf, New BrunswickTM, model: Innova® 4000 )
    Note: This item has been discontinued. Possible substitute: Eppendorf, New BrunswickTM, model: Innova® 40.
  6. Centrifuge 5810 R (Eppendorf, model: 5810 R , catalog number: 5811000320)
  7. Multigas incubator (SANYO, model: MCO-19M )
    Note: This item has been discontinued. Possible substitute: Panasonic Healthcare, model: MCO-170M .
  8. Laminar flow tissue culture hood*
  9. Inverted microscope*
  10. Hemocytometer*
  11. Refrigerator*
  12. Ultra-low temperature freezer (Panasonic Healthcare, catalog number: MDF-U3386S )
  13. Locator 6 Plus Rack and Box Systems, liquid nitrogen tank (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CY50985-70 )
  14. Autoclave*

*Note: This item can be ordered from any qualified company.

Procedure

  1. Isolation of murine MSCs
    Note: Perform all manipulations under sterile conditions. In order to keep digestion conditions reproducible between subsequent cell isolations, the volumes of solutions during the crucial collagenase digestion step are determined based on the weight of adipose tissue.
    1. Sacrifice mice by cervical dislocation. Sterilize animals by submerging into 70% ethanol for 5-10 min.
      Note: Mice may also be euthanized by CO2 inhalation or other approved methods. The number of animals used for isolation depends on the set goals. Given the high expansion capacity of MSCs, one to two animals would suffice for most applications.
    2. Dissect the abdominal skin to expose subcutaneous adipose tissue (Video 1).

      Video 1. Mouse dissection

    3. Extract the subcutaneous adipose tissue using sterile forceps and scissors (Video 2).

      Video 2. Excision of adipose tissue

    4. Place the extracted tissue to a pre-weighted 6-cm Petri dish. Measure the weight of extracted tissue (N g) using an analytical balance.
    5. Add 1x N ml (where N is the weight of adipose tissue in grams) of the room temperature MSC isolation medium (see Recipes) to the Petri dish containing adipose tissue.
      Note: Although adipose tissue can stay for some time in MSC isolation medium until collagenase solution (see Recipes) for tissue dissociation is added, it is recommended to recruit an assistant solely for this step, whose task would be to prepare the collagenase solution once the weight of extracted adipose tissue is determined.
    6. Mince adipose tissue with sterile scissors until a fine-pieced slurry is formed (Video 3).

      Video 3. Mincing of adipose tissue

    7. Using a 1-ml filter tip with the cut-off end, transfer accurately the minced tissue to a 15-ml centrifuge tube (Video 3).
      Note: If the weight of adipose tissue exceeds 2 g, use a 50-ml centrifuge tube instead.
    8. Add 2.5x N ml of MSC isolation medium to the Petri dish, wash and transfer the remaining pieces of tissue to the same 15-ml tube.
    9. Add 0.5x N ml of the prepared collagenase solution to the tube with the minced tissue (Video 3).
    10. Incubate for 40 min at 37 °C with constant agitation in a thermostated shaker at a speed of 200 RPM (Video 4).

      Video 4. Treatment with collagenase and centrifugation

    11. Pipette up and down the resulting cell suspension 20-30 times using a 10 ml pipette until homogeneous.
    12. Centrifuge cell suspension at 22 °C for 10 min at 370 x g (Video 4).
    13. Discard the supernatant, add 1 ml of MSC isolation medium and resuspend cells using a 1-ml filter tip. Add 9 ml of MSC isolation medium, mix and centrifuge at 22 °C for 10 min at 370 x g (Video 4).
    14. Repeat the step A12 once more. Resuspend cells in 1 ml of MSC growth medium (see Recipes).
    15. Count the total number of isolated cells using hemocytometer.
      Note: The average number of isolated cells at this stage is variable but usually about 5-15 million cells per mouse.
    16. Seed 5 to 7 million cells per 10-cm Petri dish, add MSC growth medium to each dish to a final volume of 10 ml, and cultivate at 37 °C in a CO2 incubator under hypoxic conditions (5% CO2, 5% O2) (Video 4).
      Note: When adding MSC growth medium, aim to spread cells evenly throughout the dish by tilting it and/or using pipet.
    17. Culture passage zero cells for 5-7 days up to 70-80% confluency, with MSC growth medium changes after the first day and every 3 days afterwards. The cells at this stage have a slightly elongated fibroblast-like shape.
    18. Tilt the Petri dish and carefully aspirate the culture medium from the plastic-attached cells avoiding their disturbance and detachment, wash once with 1x PBS (see Recipes).
    19. Add 1 ml of trypsin solution (see Recipes) to each 10-cm Petri dish, detach cells at 37 °C for 5-10 min.
      Note: Monitor the degree of cell detachment microscopically after 5 min of incubation; if necessary, extend incubation until the vast majority of cells are detached, but not longer than 10 min (see Note 3).
    20. Transfer cells to a 15-ml centrifuge tube, add 5 ml of MSC growth medium, mix by gentle pipetting and centrifuge at room temperature for 7 min at 370 x g.
    21. Suspend cells in 1-2 ml of MSC growth medium.
    22. Mix a cell aliquot with an equal volume of trypan blue solution and count live cells using hemocytometer.

  2. Expansion of murine MSCs
    Note: Perform all manipulations under sterile conditions.
    1. Seed the MSC cell suspension into appropriate culture vessels at a density of 200 to 400 cells/cm2.
    2. Culture cells in MSC growth medium for 3 to 4 days in a CO2 incubator under hypoxic conditions (5% CO2, 5% O2) until reaching 50% to 70% confluency. Change medium every 3 days.
      Notes:
      1.  The effect of hypoxic conditions on murine MSC growth is illustrated in the Figure 1.
      2. The optimal culture time depends on seeding density: for 200 cells/cm2 the optimal culture time is usually 4 days, whereas for 400 cells/cm2–3 days.
    3. Detach cells with trypsin and count them analogously to the steps A16-A20 of the Procedure A above.
    4. Repeat steps B1-B3 until the required number of MSC is obtained.


      Figure 1. Expansion of mouse adipose tissue MSCs under hypoxia (5% O2) and normoxia conditions. Cells were seeded at a density of 200 cells/cm2 in 3.5-cm Petri dishes and grown in MSC growth medium for 4 days followed by the transfer to the next passage. Y axis depicts the magnitude of expansion factor, defined as a ratio of a number of live cells collected at the end of the passage to the number of seeded live cells. Data are presented as mean ± SD. Cells grown at normoxia show lower growth rates and faster proliferation decline within 4 passages as compared to the cells grown under hypoxia conditions. Importantly, mouse MSCs at normoxia do not proliferate longer than 3-4 weeks whereas those at hypoxia can proliferate for at least 8-9 weeks before becoming senescent.

  3. Freezing MSCs
    Note: Perform all manipulations under sterile conditions. The protocol below is intended for freezing 4-5 million cells (8-10 frozen cell samples). If necessary, scale up or down the protocol to accommodate for desired cell numbers.
    1. Grow MSCs in six 10-cm Petri dishes under hypoxia conditions until reaching 70% to 80% confluency.
    2. Detach cells with 1 ml of trypsin solution per dish as described above.
    3. Transfer cells to a 15-ml centrifuge tube, add 5 ml of MSC growth medium, mix with pipette and centrifuge at room temperature for 7 min at 370 x g.
    4. Discard the supernatant and resuspend cells in 4 ml of growth medium.
    5. Count the total number of live cells using hemocytometer as described in the step A22 of the Procedure A.
      Note: The total number of collected cells is expected to be about 4-5 x 106.
    6. Add to the cells 5 ml of fetal calf serum.
    7. Add to the cells 1 ml of DMSO. Addition should be performed dropwise to the cells kept under constant agitation (vortexing).
      Note: After addition of DMSO, subsequent operations with cells should be performed promptly as high concentrations of DMSO are harmful for cells.
    8. Aliquot 1.0 ml of cell suspension into pre-labeled cryotubes (8-10 cryotubes in total).
    9. Place the cryotubes into a box lined with cotton wool and transfer to a -80 °C freezer for 2-3 days.
    10. Transfer the cryotubes into a liquid nitrogen tank.

  4. Thawing MSCs
    Note: Perform all manipulations under sterile conditions. The protocol below is designed for thawing one frozen cell sample. If necessary, scale up the protocol to accommodate for desired cell numbers.
    1. Remove the cryotube with frozen MSCs from the liquid nitrogen tank, let cell suspension thaw at room temperature.
      Note: Make sure cells are completely thawed within 10 min. Hand warming may be used to speed up the process.
    2. Add 10 ml of MSC growth medium to the 15-ml centrifuge tube and transfer thawed cells to the same tube.
    3. Centrifuge cells at room temperature for 7 min at 370 x g.
    4. Carefully aspirate the supernatant using the pipet, suspend cells in 1 ml of MSC growth medium.
    5. Count the total number of live cells using hemocytometer as described in the step A22 of the Procedure A.
      Note: The total number of live cells at this stage may vary but usually is about 250-350 thousand per one frozen cell sample.
    6. Transfer cells to a 10-cm Petri dish, add MSC growth medium to a final volume of 10 ml.
    7. Culture cells in MSC growth medium in a CO2 incubator under hypoxic conditions (5% CO2, 5% O2) until reaching 50% to 70% confluency. Change medium every 3 days.
      Note: Growth for 5 to 7 days is considered as sufficient for cells to recover and acquire normal physiological state.

Data analysis

Assessment of quality of cells isolated and expanded using the Procedure A and Procedure B is performed primarily by flow cytometry using antibodies against essential cell surface markers of mouse MSCs, namely positive surface markers CD29, CD44, CD105, and negative markers CD45, CD11b, CD34. After passage 2, the expanded MSC populations are expected to be 98-99% positive for CD29 and CD44 markers, and about 98-99% negative for CD11b, CD45 and CD34 markers, whereas they are heterogeneous for expression of CD105 (see Figure 2). Additionally, differentiation into osteocytes, adipocytes and chondrocytes (Sun et al., 2003) may be performed to demonstrate the multilineage differentiation capacity of expanded cell populations. Isolated MSC populations should demonstrate rapid growth, with 12 to 16 h population doubling times during the first 3-4 weeks in culture.
Note: Depending on conditions of analysis, the percentage of CD34-positive cells may vary and be higher than 1-2%.


Figure 2. Cell surface phenotype characterization of mouse adipose tissue MSCs by flow cytometry. Cells were stained with propidium iodide (PI), and viable (PI-negative gated) cells were analyzed for binding of fluorescently labeled antibodies against positive markers CD29 and CD44, partially positive marker CD105, weakly positive marker CD34, and negative markers CD11b and CD45.

Notes

  1. For cell isolation, female mice aged about 8 weeks are normally used.
  2. In the described protocol, removal of remaining undigested pieces of adipose tissues by filtration through cell strainers is not used as this might lead to cell entrapment and losses. The tissue pieces do not interfere with cell growth and are eventually removed during media changes.
  3. Trypsin treatment should not exceed 10 min as longer exposures may negatively affect cell state.
  4. Culture media should be warmed up to 37 °C prior to work with cells during their expansion.
  5. The described culture conditions were empirically determined by us as providing the fastest expansion rates due to significant growth retardation both at low seeding densities and under subconfluent conditions.
  6. Under optimal hypoxia culture conditions, murine MSCs can undergo about 80 population doublings before becoming senescent. Spontaneous immortalization or transformation of these cells has never been observed by us under hypoxia growth conditions.

Recipes

  1. DMEM low glucose medium (5 L)
    50 g DMEM low glucose medium powder
    18.5 g sodium hydrogen carbonate cell culture grade
    Add water to a final volume of 5 L
    Sterilize by vacuum filtration
    Store at 4 °C
    Use within 6 months
  2. MSC isolation medium (100 ml)
    93 ml DMEM low glucose medium
    5 ml penicillin-streptomycin (100x)
    2 ml amphotericin B
    Combine sterile components under the laminar flow hood
    Store at 4 °C
    Use within 1 month
  3. Collagenase solution (prepare freshly)
    Collagenase 12x N mg (where the N is the weight of adipose tissue)
    MSC isolation medium 0.6x N ml
    Sterilize by filtration through 0.22 mm filter under laminar flow hood
  4. MSC growth medium (1 L)
    880 ml DMEM low glucose medium
    100 ml fetal bovine serum
    10 ml GlutaMax (100x)
    10 ml penicillin-streptomycin (100x)
    Combine sterile components under the laminar flow hood
    Store at 4 °C
    Use within 1 month
  5. 1x phosphate-buffered saline (PBS) pH 7.4 (1 L)
    2 tablets PBS (10 g)
    Add water to a final volume of 1 L
    Sterilize by autoclaving
    Store at room temperature
  6. Trypsin solution (500 ml)
    1 tablet PBS (5 g)
    100 mg EDTA
    1.25 g trypsin
    Add water to a final volume of 500 ml
    Dissolve and sterilize by filtration
    Store at 4 °C
    Use within 6 months

Acknowledgments

The protocol described herein was adapted with modifications from Andreeva et al. (2015). This work was supported by the grant No. 17-04-02127 from the Russian Foundation for Basic Research and by the program ‘Basic research for the development of biomedical technologies’ of the Presidium of the Russian Academy of Sciences.

References

  1. Andreeva, N. V., Bonartsev, A. P., Zharkova, II, Makhina, T. K., Myshkina, V. L., Kharitonova, E. P., Voinova, V. V., Bonartseva, G. A., Shaitan, K. V. and Belyavskii, A. V. (2015). Culturing of mouse mesenchymal stem cells on Poly-3-Hydroxybutyrate scaffolds. Bull Exp Biol Med 159(4): 567-571.
  2. Boregowda, S. V., Krishnappa, V., Chambers, J. W., Lograsso, P. V., Lai, W. T., Ortiz, L. A. and Phinney, D. G. (2012). Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion. Stem Cells 30(5): 975-987.
  3. Caplan, A. I. (2016). MSCs: The sentinel and safe-guards of injury. J Cell Physiol 231(7): 1413-1416.
  4. Friedenstein, A. J., Gorskaja, J. F. and Kulagina, N. N. (1976). Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 4(5): 267-274.
  5. Krishnappa, V., Boregowda, S. V. and Phinney, D. G. (2013). The peculiar biology of mouse mesenchymal stromal cells--oxygen is the key. Cytotherapy 15(5): 536-541.
  6. Murray, I. R., West, C. C., Hardy, W. R., James, A. W., Park, T. S., Nguyen, A., Tawonsawatruk, T., Lazzari, L., Soo, C. and Peault, B. (2014). Natural history of mesenchymal stem cells, from vessel walls to culture vessels. Cell Mol Life Sci 71(8): 1353-1374.
  7. Phinney, D. G. and Sensebe, L. (2013). Mesenchymal stromal cells: misconceptions and evolving concepts. Cytotherapy 15(2): 140-145.
  8. Prockop, D. J. (2013). Concise review: two negative feedback loops place mesenchymal stem/stromal cells at the center of early regulators of inflammation. Stem Cells 31(10): 2042-2046.
  9. Sun, S., Guo, Z., Xiao, X., Liu, B., Liu, X., Tang, P. H. and Mao, N. (2003). Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells 21(5): 527-535.

简介

间充质干细胞(MSC)目前正在深入研究,因为它们代表未来细胞治疗临床方案的成功实施的重大前景。 这又强调了对各种鼠疾病模型中MSC效应的仔细临床前研究的重要性。 具有可重现的生物学性质的合适的细胞制剂对于最小化实验细胞疗法结果的变异性是重要的。 我们在这里描述了一种用于从脂肪组织中分离鼠MSC的简单方案及其在缺氧条件下的可重复的多对数扩增。
【背景】最初由Friedenstein鉴定的MSC是成纤维细胞样形态的骨髓细胞,粘附于塑料和高自我更新能力,导致体外成纤维细胞样集落的形成(Friedenstein等,1976; Review in Phinney andSensebé ,2013)。 MSCs由于其在医学上的潜在应用,目前是研究最成熟的成体祖细胞类型之一。这些细胞可以从各种器官中分离(Murray等,2014),并且被认为是源于血管,以周细胞或血管壁细胞。除了能够沿着成骨,脂肪形成和软骨形成谱系分化的能力之外,MSC具有免疫调节特性,并且被认为参与对组织损伤的反应,以及通过其影响巨噬细胞极化的能力来组织抗炎反应(Prockop,2013; Caplan,2016)。
   鉴于这些特性,MSCs代表了未来相关细胞治疗临床方案的成功实施的巨大前景。这反过来强调了在各种鼠疾病模型中使用MSC进行仔细临床前研究的重要性。制备大量具有可重复生物学特性的合适细胞样品的能力对于在开发基于MSC的实验细胞疗法期间最小化结果的变异性至关重要。然而,与具有强抗氧化防御性并因此在大气氧条件下相当好的人类MSC不同,小鼠MSC对氧应激更敏感,并且在常规CO2培养箱中培养时具有有限的寿命和扩张能力。相反,在缺氧条件下培养这些细胞,相反,显着延长了它们的寿命,并允许多对数扩增,提供足够量的具有可重复性质的细胞材料,用于用鼠实验疾病模型重复实验(Boregowda等,2012; Krishnappa et al。等等,2013)。在本文中,我们描述了从脂肪组织中分离鼠MSC的简单方案,其在缺氧条件下的可重复扩增以及长期储存。

关键字:间充质干细胞, 脂肪组织, 氧化应激, 缺氧, 扩增

材料和试剂

  1. 6厘米细胞培养皿(Greiner Bio One International,目录号:628160)
  2. 无菌移液器过滤嘴20μl(Greiner Bio One International,目录号:774288)
  3. 无菌移液器过滤嘴200μl(Greiner Bio One International,目录号:739288)
  4. 无菌移液器过滤嘴1000μl(Greiner Bio One International,目录号:740288)
  5. 15ml离心管(Greiner Bio One International,目录号:188261)
  6. 50ml离心管(Greiner Bio One International,目录号:227261)
  7. 1.8毫升圆底低温管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:375418)
  8. 10厘米细胞培养皿(Greiner Bio One International,目录号:664160)
  9. 3.5厘米细胞培养皿(Greiner Bio One International,目录号:627160)
  10. 棉羊毛
  11. 真空过滤器/储存瓶系统,0.22μm,500ml(Corning,目录号:431097)
  12. 0.22μm注射器过滤器(Sigma-Aldrich,目录号:Z741948)
    制造商:GVS,目录号:1214220。
  13. 10 ml注射器(SFM医院产品,目录号:534235)
  14. 2 ml血清移液管(Greiner Bio One International,目录号:710180)
  15. 5 ml血清移液管(Greiner Bio One International,目录号:606180)
  16. 10 ml血清移液管(Greiner Bio One International,目录号:607107)
  17. 25 ml血清移液管(Greiner Bio One International,目录号:760160)
  18. C57BL / 6小鼠
  19. 无菌蒸馏水
  20. 乙醇96%(Sigma-Aldrich,目录号:24105)
    注意:本产品已停产。
  21. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  22. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  23. 碘化丙啶(PI)(Thermo Fisher Scientific,Invitrogen TM,目录号:P3566)
  24. 细胞培养基成分
    1. DMEM低葡萄糖,粉末(Thermo Fisher Scientific,Gibco TM,目录号:31600083)
    2. GlutaMax(100x)(Thermo Fisher Scientific,目录号:35050038)
    3. 青霉素 - 链霉素(100x)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    4. 碳酸氢钠细胞培养级(AppliChem,目录号:A0384)
  25. 两性霉素B(0.25mg / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15290018)
  26. 来自<溶解性Clostridium histolyticum 的胶原酶(Sigma-Aldrich,目录号:C2674)
  27. 胎牛血清(GE Healthcare,HyClone TM,目录号:SV30160.03)
  28. 磷酸缓冲盐水(PBS),pH 7.4片(Thermo Fisher Scientific,目录号:18912014)
  29. 来自猪胰腺的胰蛋白酶(Sigma-Aldrich,目录号:T4799)
  30. 乙二胺四乙酸二钠盐(EDTA)(Sigma-Aldrich,目录号:E5134)
  31. DMEM低葡萄糖培养基(参见食谱)
  32. MSC隔离介质(见配方)
  33. 胶原酶溶液(参见食谱)
  34. MSC生长培养基(见食谱)
  35. 1x磷酸缓冲盐水(PBS)pH 7.4(参见食谱)
  36. 胰蛋白酶溶液(参见食谱)

设备

  1. 消毒手术工具包括镊子和剪刀
  2. 移液器控制器(Corning,目录号:4091)
  3. 自动单通道移液器,0.5-20,20-200和100-1,000μl,Gilson兼容(Gilson)
  4. 分析天平*
  5. 恒温摇床(Eppendorf,New Brunswick TM ,型号:Innova ® 4000)
    注意:此项目已被停用。可能的替代品:Eppendorf,New Brunswick TM ,型号:Innova ® sup> 40。
  6. 离心机5810 R(Eppendorf,型号:5810 R,目录号:5811000320)
  7. Multigas孵化器(SANYO,型号:MCO-19M)
    注意:此项目已被停用。可能的替代品:Panasonic Healthcare,型号:MCO-170M。
  8. 层流组织培养罩*
  9. 倒置显微镜*
  10. 血细胞计数器*
  11. 冰箱*
  12. 超低温冷冻机(Panasonic Healthcare,目录号:MDF-U3386S)
  13. 定位器6 Plus机架和箱系统,液氮罐(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:CY50985-70)
  14. 高压灭菌器*

*注意:该项目可以从任何合格的公司订购。

程序

  1. 小鼠MSC的分离
    注意:在无菌条件下执行所有操作。为了保持消化条件在随后的细胞分离之间可重现,在关键胶原酶消化步骤期间溶液的体积基于脂肪组织的重量来确定。
    1. 牺牲小鼠颈椎脱位。通过浸入70%乙醇中灭菌5-10分钟来灭菌动物 注意:小鼠也可以通过CO 2 吸入或其他批准的方法进行安乐死。用于隔离的动物数量取决于设定的目标。鉴于MSC的扩张能力很强,一到两只动物对大多数应用来说都是足够的。
    2. 解剖腹部皮肤以暴露皮下脂肪组织(视频1)。

      Video 1. Mouse dissection

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    3. 使用无菌镊子和剪刀提取皮下脂肪组织(视频2)
      Video 2. Excision of adipose tissue

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    4. 将提取的组织放置在预先加重的6厘米培养皿中。 使用分析天平测量提取的组织的重量(N g)。
    5. 将室温MSC分离培养基(见食谱)的1x N ml(其中N是脂肪组织的重量)加到含有脂肪组织的培养皿中。
      注意:虽然脂肪组织可以在MSC分离培养基中停留一段时间,直到胶原酶溶液(参见食谱)用于组织解离,但建议仅为此步骤招募一名助手,其任务是制备胶原酶 确定提取的脂肪组织的重量后的溶液。
    6. 用无菌剪刀将脂肪组织切碎,直至形成精细拼接的浆液(视频3)
      Video 3. Mincing of adipose tissue

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    7. 将提取的组织放置在预先加重的6厘米培养皿中。 使用分析天平测量提取的组织的重量(N g)。
    8. 将室温MSC分离培养基(见食谱)的1x N ml(其中N是脂肪组织的重量)加到含有脂肪组织的培养皿中。
      注意:虽然脂肪组织可以在MSC分离培养基中停留一段时间,直到胶原酶溶液(参见食谱)用于组织解离,但建议仅为此步骤招募一名助手,其任务是制备胶原酶 确定提取的脂肪组织的重量后的溶液。
    9. 用无菌剪刀将脂肪组织切碎,直至形成精细拼接的浆液(视频3)
      Video 4. Treatment with collagenase and centrifugation

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    10. 使用10ml移液管将所得细胞悬浮液上下移动20-30次,直到均匀
    11. 在370℃离心细胞悬浮液在22℃下10分钟(视频4)。
    12. 丢弃上清液,加入1 ml的MSC分离培养基,并使用1 ml过滤嘴重悬细胞。加入9 ml的MSC分离培养基,混合并在370℃离心22分钟10分钟(视频4)。
    13. 再次重复步骤A12。将细胞重悬于1ml的MSC生长培养基中(见食谱)
    14. 使用血细胞计数器计数分离细胞的总数。
      注意:在这个阶段,孤立细胞的平均数量是可变的,但通常每只小鼠约5-15万个细胞。
    15. 每10厘米培养皿种子5至700万个细胞,每个培养皿加入MSC生长培养基至终体积为10ml,并在缺氧条件下于CO 2培养箱中于37℃培养( 5%CO 2,5%O 2)(视频4)。
      注意:当添加MSC生长培养基时,目的是通过倾斜和/或使用移液管将细胞均匀分布在整个盘中。
    16. 文化通道零细胞5-7天达70-80%融合,MSC生长培养基在第一天和每3天后变化。在这个阶段的细胞有一个稍长的成纤维细胞样的形状
    17. 倾斜培养皿,并从塑料连接的细胞中小心地吸出培养基,避免其干扰和分离,用1x PBS洗涤一次(参见食谱)。
    18. 向每个10 cm培养皿中加入1 ml胰蛋白酶溶液(见食谱),37℃下分离细胞5-10分钟。
      注意:孵育5分钟后,显微镜观察细胞脱离程度;如果需要,延长孵化,直到绝大多数细胞分离,但不超过10分钟(见注3)。
    19. 将细胞转移到15ml离心管中,加入5ml MSC生长培养基,通过轻轻移液混合并在370×g下室温离心7分钟。
    20. 将细胞悬浮于1-2ml的MSC生长培养基中。
    21. 用等体积的台盼蓝溶液混合细胞等分试样,并使用血细胞计数器计数活细胞
  2. 扩大小鼠MSC /
    注意:在无菌条件下执行所有操作。
    1. 将MSC细胞悬浮液以200至400个细胞/ cm 2的密度将MSC细胞接种到合适的培养容器中。
    2. 在低氧条件(5%CO 2,5%O 2)的CO 2培养箱中,在MSC生长培养基中培养细胞3至4天, )直至达到50%至70%汇合。每3天换一次媒体。
      注意:
      1. 缺氧条件对小鼠MSC生长的影响如图1所示。
      2. 最佳培养时间取决于播种密度:对于200个细胞/ cm 2,最佳培养时间通常为4天,而对于400个细胞/ cm 3 -3天。 />
    3. 用胰蛋白酶分离细胞,并按照上述步骤A的步骤A16-A20进行计数。
    4. 重复步骤B1-B3,直到获得所需数量的MSC。


      图1.小鼠脂肪组织在缺氧(5%O <2> )和常氧条件下的扩增细胞在3.5cm培养皿中以200个细胞/ cm 2的密度接种,并在MSC生长培养基中培养4天,然后转移到下一道。 Y轴描绘了膨胀因子的大小,其定义为在通道结束时收集的活细胞数与接种活细胞数量的比率。数据以平均值±SD表示。与在缺氧条件下生长的细胞相比,在正常氧条件下生长的细胞在4代中显示较低的生长速率和更快的增殖下降。重要的是,正常氧中的小鼠MSC不会增长超过3-4周,而缺氧的小鼠可以在衰老前增殖至少8-9周。

  3. 冻结MSCs
    注意:在无菌条件下执行所有操作。以下方案用于冻结4-5百万个细胞(8-10个冷冻细胞样品)。如果需要,按照协议扩大或缩小以适应所需的单元格号。
    1. 在缺氧条件下,在六个10厘米培养皿中培养MSC,直至达到70%至80%的汇合度。
    2. 如上所述,每个皿用1ml胰蛋白酶溶液分离细胞。
    3. 将细胞转移到15 ml离心管中,加入5ml MSC生长培养基,用移液管混合,室温下离心3分钟,速度为370 x g。
    4. 弃去上清液,将细胞重悬于4 ml生长培养基中
    5. 使用血细胞计数器计数活细胞的总数,如步骤A的步骤A22所述 注意:收集的单元格的总数预计约为4-5 x 10
      />
    6. 加入细胞5ml 5ml胎牛血清
    7. 向细胞中加入1ml DMSO。应该向保持恒定搅拌(涡旋)的细胞滴加 注意:加入DMSO后,由于高浓度的DMSO对细胞有害,因此应及时进行细胞随后的手术。
    8. 将1.0ml细胞悬浮液等分成预先标记的冷冻管(总共8-10个冻管)。
    9. 将低温管放入衬有棉绒的箱子中,并转移到-80°C的冷冻箱中2-3天。
    10. 将冷冻管转移到液氮罐中
  4. 解冻MSC
    注意:在无菌条件下执行所有操作。下面的方案设计用于解冻一个冷冻细胞样品。如有必要,可以扩展协议以适应所需的单元格号。
    1. 用冷冻的MSCs从液氮罐中取出冷冻管,让细胞在室温下解冻 注意:确保细胞在10分钟内完全解冻。手工升温可用于加快过程。
    2. 将10 ml的MSC生长培养基加入到15 ml离心管中,并将解冻的细胞转移到同一管中
    3. 在370×g下室温离心细胞7分钟。
    4. 使用移液管小心吸出上清液,将细胞悬浮于1ml的MSC生长培养基中
    5. 使用血细胞计数器计数活细胞的总数,如步骤A的步骤A22所述 注意:此阶段的活细胞总数可能有所不同,但通常每个冻结细胞样本约为250-350千克。
    6. 将细胞转移到10厘米培养皿中,加入MSC生长培养基至终体积为10毫升
    7. 在低氧条件(5%CO 2,5%O 2)中的CO 2培养箱中的MSC生长培养基中培养细胞,直至达到50%至70%汇合。每3天换一次媒体。
      注意:5至7天的生长被认为足以使细胞恢复并获得正常的生理状态。

数据分析

使用方法A和方法B分离和扩增的细胞的质量评估主要通过使用针对小鼠MSC的基本细胞表面标志物的抗体的流式细胞术进行进行,即阳性表面标志物CD29,CD44,CD105和阴性标记物CD45,CD11b,CD34 。第2代后,扩增的MSC群体对于CD29和CD44标记预期为98-99%阳性,CD11b,CD45和CD34标记为98-99%为阴性,而CD105表达则为异质性(见图2 )。此外,可以进行分化为骨细胞,脂肪细胞和软骨细胞(Sun等人,2003),以证明扩增细胞群体的多谱系分化能力。孤立的MSC群体应显示出快速生长,培养前3-4周的群体繁殖时间为12至16 h 注意:根据分析条件,CD34阳性细胞的百分比可能会变化并高于1-2%。


图2.通过流式细胞术检测小鼠脂肪组织MSC的细胞表面表型细胞用碘化丙啶(PI)染色,分析活细胞(PI阴性门控)细胞结合荧光标记的抗体针对阳性标志物CD29和CD44,部分阳性标记物CD105,弱阳性标志物CD34和阴性标志物CD11b和CD45。

笔记

  1. 对于细胞分离,通常使用约8周龄的雌性小鼠
  2. 在所述方案中,不使用通过细胞过滤器过滤除去剩余的未消化的脂肪组织块,因为这可能导致细胞的捕获和损失。组织块不会干扰细胞生长,并且最终在培养基更换期间被去除
  3. 胰蛋白酶处理不应超过10分钟,因为较长的暴露可能会对细胞状态产生负面影响
  4. 在培养基扩张前,应将细胞培养至37°C
  5. 所描述的培养条件由我们经验确定,因为在低播种密度和亚融合条件下由于显着的生长迟缓而提供最快的扩张率。
  6. 在最佳缺氧培养条件下,小鼠MSC可以在衰老之前经历大约80次群体倍增。我们在缺氧生长条件下从未观察到这些细胞的自发永生化或转化

食谱

  1. DMEM低葡萄糖培养基(5升)
    50克DMEM低葡萄糖培养基粉末 18.5g碳酸氢钠细胞培养等级
    加水至最终体积5升
    真空过滤灭菌
    储存于4°C
    6个月内使用
  2. MSC隔离培养基(100 ml)
    93毫升DMEM低葡萄糖培养基 5 ml青霉素 - 链霉素(100x)
    2 ml两性霉素B
    在层流罩下组合无菌组件
    储存于4°C
    1个月内使用
  3. 胶原酶溶液(新鲜准备)
    胶原酶12x N mg(其中N是脂肪组织的重量)
    MSC隔离介质0.6x N ml
    在层流罩下通过0.22mm过滤器过滤灭菌
  4. MSC生长培养基(1L)
    880毫升DMEM低葡萄糖培养基 100毫升胎牛血清
    10 ml GlutaMax(100x)
    10 ml青霉素 - 链霉素(100x)
    在层流罩下组合无菌组件
    储存于4°C
    1个月内使用
  5. 1x磷酸缓冲盐水(PBS)pH 7.4(1L)
    2片PBS(10克)
    加水至最终体积为1升
    高压消毒灭菌
    在室温下存放
  6. 胰蛋白酶溶液(500毫升)
    1片PBS(5克)
    100毫克EDTA
    1.25克胰蛋白酶
    加水至最终体积为500 ml
    通过过滤溶解和灭菌
    储存于4°C
    6个月内使用

致谢

本文描述的方案适应于Andreeva等人(2015)的修改。这项工作得到俄罗斯基础研究基金会批准的第17-04-02127号和俄罗斯科学院主席团发展生物医学技术的基础研究项目的支持。

参考

  1. Andriva,NV,Bonartsev,AP,Zharkova,II,Makhina,TK,Myshkina,VL,Kharitonova,EP,Voinova,VV,Bonartseva,GA,Shaitan,KV和Belyavskii,AV(2015)。&lt; a class = ke-insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/26388561“target =”_ blank“>在聚-3-羟基丁酸酯支架上培养小鼠间充质干细胞。 Bull Exp Biol Med 159(4):567-571。
  2. Boregowda,SV,Krishnappa,V.,Chambers,JW,Lograsso,PV,Lai,WT,Ortiz,LA和Phinney,DG(2012)。&lt; a class =“ke-insertfile”href =“http: www.ncbi.nlm.nih.gov/pubmed/22367737“target =”_ blank“>大气氧通过p53依赖性机制抑制骨髓来源的小鼠间充质干细胞的生长和分化:对长期培养扩增的影响。 / a>干细胞 30(5):975-987。
  3. Caplan,AI(2016)。 MSC:哨兵和安全防护装置。 J Cell Physiol 231(7):1413-1416。
  4. Friedenstein,AJ,Gorskaja,JF和Kulagina,NN(1976)。&nbsp;
  5. Krishnappa,V.,Boregowda,SV和Phinney,DG(2013)。&nbsp; 小鼠间充质基质细胞的特殊生物学 - 氧气是关键。细胞疗法 15(5):536-541。
  6. Murray,IR,West,CC,Hardy,WR,James,AW,Park,TS,Nguyen,A.,Tawonsawatruk,T.,Lazzari,L.,Soo,C.and Peault,B。(2014) 间质干细胞的自然史,从血管壁到培养血管。细胞分子生命科学 71(8):1353-1374。
  7. Phinney,DG和Sensebe,L。(2013)。间质基质细胞:误解和进化概念。细胞疗法 15(2):140-145。
  8. Prockop,DJ(2013)。简明回顾:两个负面反馈回路将间质干细胞/基质细胞置于早期炎症调节因子的中心。 31(10):2042-2046。
  9. Sun,S.,Guo,Z.,Xiao,X.,Liu,B.,Liu,X.,Tang,PH and Mao,N。(2003)。通过新颖可靠的方法分离小鼠骨髓间充质祖细胞。干细胞 21(5):527-535。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Andreeva, N. V., Dalina, A. and Belyavsky, A. V. (2017). Isolation and Expansion of Mesenchymal Stem Cells from Murine Adipose Tissue. Bio-protocol 7(16): e2516. DOI: 10.21769/BioProtoc.2516.
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