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Mesenchymal stromal cells (MSCs) are non-hematopoietic, perivascular cells which support hematopoiesis and are thought to participate in tissue repair in vivo. MSCs can be isolated from various tissues and organs and are defined in vitro as plastic adherent cells expressing CD73, CD90, CD105 (human MSCs) or CD29, CD44, sca-1 (murine MSCs) which can differentiate into osteoblasts, adipocytes, chondroblasts and myocytes. MSCs possess potent immunomodulatory and trophic capacities in vitro and in vivo and have thus emerged as a promising treatment of inflammatory/autoimmune diseases. The use of MSCs for human disease relies on the injection of a large number of cells and much effort has been focused on acquiring MSCs with high proliferative capacity. Thus, establishing simple and accurate protocols for measuring MSC proliferation is of importance for both basic and applied research. The current protocol provides details on how to isolate and measure the proliferation of murine MSCs derived from inguinal and/or intraabdominal adipose tissue (mASCs) using the xCELLigence system and CellTiter-Blue reagent (Carrillo-Galvez et al., 2015; Anderson et al., 2013). The protocols described below can also be easily translated to human MSCs.

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Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro
鼠脂肪组织源间充质基质细胞(mASC)的分离和体外增殖分析

干细胞 > 成体干细胞 > 造血干细胞
作者: Per Anderson
Per AndersonAffiliation: Genomic Oncology Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalucian Regional Government, PTS Granada, Granada, Spain
For correspondence: per.anderson@genyo.es
Bio-protocol author page: a2690
Ana Belén Carrillo-Gálvez
Ana Belén Carrillo-GálvezAffiliation: Genomic Oncology Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-University of Granada-Andalucian Regional Government, PTS Granada, Granada, Spain
Bio-protocol author page: a2691
 and Francisco Martín
Francisco MartínAffiliation: Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalucian Regional Government, PTS Granada, Granada, Spain
Bio-protocol author page: a2692
Vol 5, Iss 21, 11/5/2015, 1406 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1642

[Abstract] Mesenchymal stromal cells (MSCs) are non-hematopoietic, perivascular cells which support hematopoiesis and are thought to participate in tissue repair in vivo. MSCs can be isolated from various tissues and organs and are defined in vitro as plastic adherent cells expressing CD73, CD90, CD105 (human MSCs) or CD29, CD44, sca-1 (murine MSCs) which can differentiate into osteoblasts, adipocytes, chondroblasts and myocytes. MSCs possess potent immunomodulatory and trophic capacities in vitro and in vivo and have thus emerged as a promising treatment of inflammatory/autoimmune diseases. The use of MSCs for human disease relies on the injection of a large number of cells and much effort has been focused on acquiring MSCs with high proliferative capacity. Thus, establishing simple and accurate protocols for measuring MSC proliferation is of importance for both basic and applied research. The current protocol provides details on how to isolate and measure the proliferation of murine MSCs derived from inguinal and/or intraabdominal adipose tissue (mASCs) using the xCELLigence system and CellTiter-Blue reagent (Carrillo-Galvez et al., 2015; Anderson et al., 2013). The protocols described below can also be easily translated to human MSCs.

[Abstract]

Materials and Reagents

  1. Scalpel blades no. 24 (VWR International, catalog number: 233-5487 )
  2. Cell strainers (100 μm and 70 μm) (BD Biosciences, catalog number: 352360 and 352350 )
    Note: Currently, it is “Corning , catalog number: 352360 and 352350”.
  3. Black 96-well plate (Greiner Bio-One GmbH, catalog number: 655076 )
  4. 16-well E-plate (E-plate 16) (ACEA BIO, catalog number: 0 6465382001 )
  5. Tissue culture treated Falcon 96-well plates (BD Biosciences, catalog number 353072 )
    Note: Currently, it is “Corning, catalog number: 353072”.
  6. NuncTM Cell Culture Treated EasYFlasksTM (Thermo Fisher Scientific, catalog number: 156499 )
  7. BALB/c (or any strain of choice) 8-12 weeks old male or female mice (Charles River Laboratories)
  8. Hank’s balanced salt solution (HBSS) w/ Calcium w/ Magnesium (Biowest, catalog number: L0612-500 )
  9. Collagenase Type I (Sigma-Aldrich, catalog number: C0130-100MG )
  10. MesenCultTM Proliferation Kit with MesenPureTM (Mouse) (STEMCELL Technologies, catalog number: 05512 )
  11. Penicillin-Streptomycin 10,000 U/ml (Life Technologies, Gibco, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 15140-122”.
  12. Stable Glutamine 200 mM (Biowest, catalog number: X0551-100 )
  13. DPBS w/o Calcium w/o Magnesium (Biowest, catalog number: L0615-500 )
  14. TrypLETM Express Enzyme (1x) (Life Technologies, catalog number: 12604021 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM catalog number: 12604021”.
  15. Fetal Bovine Serum (FBS) (Thermo Fisher Scientific, catalog number: 10309433 )
  16. Trypan blue (Sigma-Aldrich, catalog number: T8154-20 ml )
  17. CellTiter-Blue reagent (Promega Corporation, catalog number: G8081 )
  18. Collagenase Type I (see Recipes)
  19. Complete MesenCult medium (see Recipes)

Equipment

  1. Sterile stainless steel scissors and forceps
  2. Neubauer cell counting chamber (VWR International, catalog number: 631-1111 )
  3. Cell culture incubator (THERMO, model: HEPA class 100 )
  4. Phase contrast microscope (OLYMPUS, model: CKX41 )
  5. Centrifuge for cells (Eppendorf, model: 5810R )
  6. xCELLigence RTCA station and control unit (lap top) with the RTCA Software (ACEA BIO /ROCHE)
  7. Fluorometro [Glomax multidetection system (Promega Corporation) or equivalent]

Procedure

  1. Isolation of mASCs
    Mesenchymal stromal cells are isolated from inguinal (subcutaneous) and gonadal (intraabdominal fat deposits attached to the epididymis/testes in male mice and ovaries/uterus in female mice) fat pads.
    1. Sacrifice the mice by cervical dislocation and aseptically remove fat tissue and collect it in a 50 ml tube containing 10 ml of ice cold HBSS (Note 1).
    2. Wash the fat tissue twice in 10 ml HBSS containing 2% (v/v) of penicillin/streptomycin at room temperature (RT) and transfer the fat to a Petri dish. Remove as much liquid as possible and weigh the fat. Cut the fat tissue using sterile scissors or scalpel blades into small pieces (≤ 3 mm3).
    3. Resuspend the minced fat tissue in 2.5 ml HBSS containing 2 mg/ml collagenase Type I (equivalent to 1-5 FALGPA units and ≥ 625 collagen digestion units (CDU)/g of fat tissue) and transfer the mixture to a 15 ml tube (< 5 ml) or 50 ml tube (≥ 5 ml). Place the tube in a 37 °C water bath for 30 min; gently vortex tube for 10 sec every 5 min.
    4. Add 10 ml of HBSS to the digested fat solution and filter it using 100 μm cell strainers (Note 2).
    5. Spin down the released cells (300 x g, 7 min at 4 °C), aspirate the supernatant (Note 3) and resuspend the pellet in 10 ml HBSS.
    6. Filter the cell suspension using a 70 μm cell strainer and pellet the cells by centrifugation (300 x g, 7 min at 4 °C).
    7.  Resuspend the pellet in 2 ml complete Mesencult medium and count cells using Trypan blue. We generally obtain 0.25 ± 0.03 g fat/mouse (mean ± SEM, n = 6) and 1.9 ± 0.4 x 106 cells/g fat (mean ± SEM, n = 11).
    8. Plate the released cells in tissue culture-treated Nunc EasYFlasks at 20-30,000 cells/cm2 in complete Mesencult medium.
    9. After 24 h, change the medium in order to remove non-attached cells and debris.

  2. Cell culture
    Culture the adherent cells using complete Mesencult at 37 °C, 5% CO2, 21% O2, 90% relative humidity (RH) in 75 cm2 (T75) Nunc EasYFlask at 10,000 cells/cm2.
    1. When cultures reach 80-90% confluency, wash the cells twice with 10 ml PBS.
    2. Add 2 ml TrypLE/T75 flask and incubate at 37 °C for 5 min. Tap firmly on the side of the bottle in order to detach the cells and then add 8 ml of PBS containing 2% FBS.
    3. Resuspend the cells gently until obtaining a homogenous cell suspension and spin down the cells at 300 x g for 5 min.
    4. Count the cells as described below and seed the cells at 10,000 cells/cm2 and expand the culture until it reaches 80-90% confluency. mASCs can be used at passage 3-7 (Note 4).
    5. The phenotype of passage 3 mASCs from Balb/c mice, as measured by flow cytometry, is CD11b- (0.6 ± 0.2%), CD31- (0%), CD45- (0.03 ± 0.06%), sca-1+ (80.3 ± 14.5%), CD29+ (99.9 ± 0.1%) and CD44+ (99.8 ± 0.2%). The expression levels are shown as % of the population expressing the marker relative to the corresponding isotype control staining (mean ± standard deviation, n = 3). The expression range can vary between mouse strains. Differentiation of murine MSCs into osteoblasts, adipocytes and chondroblasts (Anderson et al., 2013; Peister et al., 2004) can be performed using commercial differentiation kits from e.g. Stem Cell Technologies (http://www.stemcell.com) and Lonza (http://www.lonza.com).

  3. Proliferation (Note 5)
    1. Preparing the mASCs (passage 3-7) for the proliferation assays.
      1. Harvest the cells as described above.
      2.  Resuspend the cell pellet gently but thoroughly in 2 ml Mesencult/confluent T75 flask to obtain a cell concentration between 5 x 105 and 1 x 106 cells/ml.
      3. Dilute the cell suspension 1:1 in Trypan blue (20 μl cell suspension + 20 μl Trypan blue).
      4. Count. Mix well and add 10 μl to a Neubauer counting chamber and count the four corner squares (Note 6).
      5. Prepare a working solution of 10,000 cells/ml in complete Mesencult for measuring cell proliferation using the xCELLigence or 6,600 cells/ml for measuring cell proliferation using the CellTiter-Blue reagent and keep the tube on ice (Note 7).
    2. xCELLigence
      The xCELLigence system can be used to measure cell proliferation in real time and uses specific cell culture plates in which the well bottoms are covered with electrodes (Figure 1). Attachment of cells to the electrodes increases their impedance which is displayed as an increase in cell index. The cell index gives information about cell number, viability and morphology in each well. The xCELLigence RTCA station should be placed in a cell incubator set at 37 °C, 21% O2, 5% CO2, 90% RH.


      Figure 1. Components of the xCELLigence system.
      The left panel shows a 16-well E-plate used for measuring proliferation of adherent cells. The middle panel shows the DP RTCA station with its three integrated stations for E-plates. The right panel gives an example of how the RTCA Software visualizes cell proliferation in real time by plotting cell index vs. time.

      1. In a sterile hood, add 100 μl/well of complete Mesencult in a 16-well E-plate, put on the lid and place it on the xCELLigence RTCA station. Check the connections and the cell index readings to make sure that the 16-well E-plate is correctly installed. Leave the 16-well E-plate on the xCELLigence RTCA station for 30-45 min and then measure the background impedance in the absence of cells.
      2. Remove the E-plate from the xCELLigence RTCA station and place it in a sterile hood. Vortex the tube containing 10,000 mASCs/ml to resuspend the cells and carefully add 100 μl of cell suspension (prepared in step C1e)/well in duplicates being careful not to touch the bottom of the wells with the tips. The final volume/well should now be 200 μl.
      3. Leave the mASCs in the xCELLigence RTCA station in the cell incubator for 7 days, reading the impedance every hour (Note 8).
      4. Plot the cell index versus time in culture (see Figure 2 below).
    3. CellTiter-Blue
      This method is based on the conversion of resazurin to the fluorescent product resorufin by metabolically active cells. The fluorescence signal obtained is proportional to the number of viable cells in the culture.
      1. Add 150 μl of cell suspension/well in a flat-bottomed 96-well tissue culture plate (each well will contain approximately 1,000 cells) in triplicates. Make 1 plate/time point. Add culture medium alone in triplicates to each plate to serve as background control. Place the plates in a cell incubator set at 37 °C, 5% CO2, 21% O2, 90% RH.
      2. Read the plates on day 1, 3 and 5-6. Add 30 μl of CellTiter-Blue reagent/well, tap the sides of the plate gently and incubate the plate for another 4 h.
      3. Transfer 100 μl from each well to a black 96-well plate and read fluorescence in a fluorometro (a Glomax multidetection system or equivalent) at 560Ex/590Em.
      4. Subtract the average background fluorescence from sample fluorescence and plot fluorescence (proliferation) versus time (see Figure 2 below).


        Figure 2. Representative data showing the proliferation of mASCs using the xCELLigence system (left panel) and the CellTiter-Blue reagent (right panel)

Notes

  1. Thoroughly spray the abdomen of the sacrificed mouse with 70% ethanol and secure the animal, ventral side up, on a polystyrene support using medical syringe needles. Use one set of sterile scissors/forceps to carefully make a midline incision, being careful not to damage the peritoneum. Separate the skin from the peritoneum and secure the skin flaps with syringe needles. When acquiring the inguinal fat pads care should be taken to remove the inguinal lymph nodes that are embedded into the fat at the Y-shaped junction of blood vessels. Using a new set of sterile scissors/forceps, collect both inguinal fat pads and transfer them to a 50 ml tube containing 10 ml HBSS and keep on ice. Proceed to cut open the peritoneum, taking care not to damage the intestines. Collect the gonadal fat pads attached to the testes and ovaries in male and female mice, respectively. We pool the fat from both locations when isolating mASCs. However, depot-specific differences in MSC phenotype have been reported and depending on your scientific aims you may have to isolate mASCs from one location or the other.
  2. The solution can be viscous and it might be necessary to use several cell strainers.
  3. The pellet in the following steps is loose and care should be taken when removing the supernatants.
  4. We seed the mASCs at a concentration of 10,000 cells/cm2 which allows us to passage the cells every 3-4 days. Under these culturing conditions we have been able to measure the proliferation of mASC from passage 3-7 using the xCELLigence system or CellTiter-Blue reagent. However, it should be noted that the size of mASCs increases with increasing passage number whereas their proliferative capacity decreases. It is therefore best to use mASC cell lines with the same passage number when comparing their proliferative capacity with the above methods.
  5. We provide two protocols for measuring mASC proliferation. The CellTiter-Blue reagent is easy to use and feasible in most laboratories. However, some studies have shown that resazurin can affect cell viability upon prolonged exposure and inclusion of drugs that increase mitochondrial number/activity can give false results. The xCELLigence system requires the purchase of the RTCA station/soft ware and the cost of specialized E-plates. However, the xCELLigence system enables the label-free and non-invasive measurement of proliferation in real-time. We generally use both methods in parallel when studying mASC proliferation in vitro. To obtain more information about the CellTiter-Blue reagent and xCELLigence system, please read the manufacturer´s instructions.
  6. We prefer to count in average 20-50 cells/corner square in order to obtain reliable cell concentrations. Calculate the cell concentration using the following formula: (total number of cells in the four corner squares/4) x 2 (dilution factor) x 10,000 = no. cells/ml.
  7. We found that the addition of 1,000 mASCs/well (3,125 cells/cm2) allows the monitoring of mASC proliferation for 5-7 days before the culture reaches confluence. This cell number gives reproducible growth curves which includes an initial 24-48 h lag phase followed by an proliferative phase according to Figure 1.
  8. We usually do not add fresh media to the E-plates during the experiment. However, it is possible to pause the experiment, remove the E-plates, add another 100 µl medium/well and restart the impedance measurements.

Recipes

  1. Collagenase Type I
    The collagenase Type I (100 mg) is dissolved in 10 ml of HBSS to a concentration of 10 mg/ml.
    Aliquots of 0.5 ml are stored at -20 °C.
  2. Complete MesenCult medium
    Thaw MesenCultTM stem cell stimulatory supplements (mouse), stable glutamine, penicillin/streptomycin at 4 °C and mix:
    400 ml of MesenCult MSC basal medium (mouse)
    100 ml MesenCult stem cell stimulatory supplements (mouse)
    5 ml stable glutamine (final concentration: 2 mM)
    5 ml penicillin/streptomycin (final concentration: 100 U/ml)
    0.5 ml Mesenpure (optional)
    Store the complete MesenCult at 4 °C and use it within one month

Acknowledgements

This work has been financed by Fondo de Investigaciones Sanitarias ISCIII (Spain) and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union, through the research grants Nº PI12/01390 (P.A), Nº PI12/01097 and ISCIII Red de Terapia Celular (TerCel: RD12/0019/0006) (F.M), by the Consejería de Innovación Ciencia y Empresa (grants Nº P09-CTS-04532 and PAIDI-Bio-326) and Consejería de Salud (grant Nº PI0001/2009) from the Junta de Andalucía and FEDER/Fondo de Cohesion Europeo (FSE) de Andalucía 2007-2013 to F.M. P.A has a Miguel Servet Contract (CP09/00228), co-financed by the Fondo Europeo de Desarollo Regional (FEDER) from the European Union. F.M. are funded by the Fundación Progreso y Salud (Consejería de Salud - Junta de Andalucía).

References

  1. Anderson, P., Carrillo-Gálvez, A. B., Garcia-Perez, A., Cobo, M. and Martín, F. (2013). CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities. PLoS One 8(10): e76979.
  2. Carrillo-Gálvez, A. B., Cobo, M., Cuevas-Ocana, S., Gutierrez-Guerrero, A., Sanchez-Gilabert, A., Bongarzone, P., Garcia-Perez, A., Munoz, P., Benabdellah, K., Toscano, M. G., Martín, F. and Anderson, P. (2015). Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity. Stem Cells 33(1): 183-195.
  3. Peister, A., Mellad, J. A., Larson, B. L., Hall, B. M., Gibson, L. F. and Prockop, D. J. (2004). Adult stem cells from bone marrow (MSCs) isolated from different strains of inbred mice vary in surface epitopes, rates of proliferation, and differentiation potential. Blood 103(5): 1662-1668.

材料和试剂

  1. 手术刀。 24(VWR International,目录号:233-5487)
  2. 细胞过滤器(100μm和70μm)(BD Biosciences,目录号:352360和352350)
    注意:目前,它是"康宁,目录号:352360和352350"。
  3. 黑色96孔板(Greiner Bio-One GmbH,目录号:655076)
  4. 16孔E板(E板16)(ACEA BIO,目录号:06465382001)
  5. 组织培养处理的Falcon 96孔板(BD Biosciences,目录号353072)
    注意:目前,它是"康宁,目录号:353072"。
  6. Nunc TM细胞培养处理的EasYFlasks TM (Thermo Fisher Scientific,目录号:156499)
  7. BALB/c(或任何选择的品系)8-12周龄雄性或雌性小鼠(Charles River Laboratories)
  8. Hank平衡盐溶液(HBSS)w /钙/镁(Biowest,目录号:L0612-500)
  9. 胶原酶I型(Sigma-Aldrich,目录号:C0130-100MG)
  10. 使用MesenPure TM sup(TM)(小鼠)(STEMCELL Technologies,目录号:05512)的MesenCult TM扩增试剂盒。
  11. 青霉素 - 链霉素10,000U/ml(Life Technologies,Gibco,目录号:15140-122)
    注意:目前,它是"Thermo Fisher Scientific,Gibco TM 目录号:15140-122"。
  12. 稳定谷氨酰胺200mM(Biowest,目录号:X0551-100)
  13. DPBS w/o Calcium w/o Magnesium(Biowest,目录号:L0615-500)
  14. TrypLE TM Express Enzyme(1x)(Life Technologies,目录号:12604021)
    注意:目前,它是"Thermo Fisher Scientific,Gibco TM 目录号:12604021"。
  15. 胎牛血清(FBS)(Thermo Fisher Scientific,目录号:10309433)
  16. 台盼蓝(Sigma-Aldrich,目录号:T8154-20ml)
  17. CellTiter-Blue试剂(Promega Corporation,目录号:G8081)
  18. I型胶原酶(参见配方)
  19. 完成MesenCult介质(参见配方)

设备

  1. 无菌不锈钢剪刀和镊子
  2. Neubauer细胞计数室(VWR International,目录号:631-1111)
  3. 细胞培养孵化器(THERMO,型号:HEPA class 100)
  4. 相差显微镜(OLYMPUS,型号:CKX41)
  5. 离心机(Eppendorf,型号:5810R)
  6. xCELLigence RTCA站和控制单元(膝上)与RTCA软件(ACEA BIO/ROCHE)
  7. Fluorometro [Glomax多检测系统(Promega Corporation)或等同物]

程序

  1. 隔离mASCs
    间充质基质细胞从腹股沟(皮下)和性腺(在雄性小鼠中附着于附睾/睾丸的腹内脂肪沉积物和卵巢/雌性小鼠的子宫)脂肪垫中分离。
    1. 通过颈部脱位牺牲小鼠,并无菌去除脂肪 组织并将其收集在含有10ml冰冷HBSS的50ml管中 (注1)。
    2. 在含有2%HBSS的10ml HBSS中洗涤脂肪组织两次, (v/v)青霉素/链霉素在室温(RT)和转移 脂肪到培养皿。尽可能多地除去液体并称重 ?脂肪。使用无菌剪刀或手术刀刀片切割脂肪组织 小块(≤3 mm 3 )。
    3. 重悬在2.5毫升的切碎的脂肪组织 含有2mg/ml I型胶原酶的HBSS(相当于1-5FALGPA 单位和≥625胶原消化单位(CDU)/g脂肪组织)和 将混合物转移到15ml管(<5ml)或50ml管(≥5ml)中。 ?将管置于37℃水浴中30分钟;轻轻涡旋管 ?每5分钟10秒。
    4. 加入10毫升HBSS消化的脂肪溶液,并使用100μm细胞过滤器过滤(注2)。
    5. 旋转释放的细胞(300×g,在4℃下7分钟),吸出 上清液(注3),并将沉淀重悬于10ml HBSS中。
    6. 使用70μm细胞过滤器过滤细胞悬浮液,并通过离心(300×g,4℃,7分钟)沉淀细胞。
    7.  在2 ml完全Mesencult培养基中重悬沉淀,计数细胞 使用台盼蓝。我们通常获得0.25±0.03g脂肪/小鼠(平均值± SEM,n = 6)和1.9±0.4×10 6个细胞/g脂肪(平均值±SEM,n = 11)。
    8. 在组织培养物处理的Nunc EasYFlasks中以20-30,000个细胞/cm 2在完全Mesencult培养基中平板释放的细胞。
    9. 24小时后,更换培养基,以去除未附着的细胞和碎片。

  2. 细胞培养
    使用完全Mesencult在37℃,5%CO 2,21%O 2,90%相对湿度(RH)在75cm 2中培养贴壁细胞(T75)Nunc EasYFlask在10,000个细胞/cm 2下。
    1. 当培养物达到80-90%汇合时,用10ml PBS洗涤细胞两次。
    2. 加入2ml TrypLE/T75烧瓶并在37℃孵育5分钟。轻按 在瓶的侧面,以便分离细胞,然后加入8ml ?的含有2%FBS的PBS。
    3. 轻轻重悬细胞,直到获得均匀的细胞悬液,并以300×g离心5分钟。
    4. 如下所述计数细胞并将细胞以10,000接种 细胞/cm 2并扩增培养物直至达到80-90%融合。 mASCs可用于3-7(注4)。
    5. 表型 来自Balb/c小鼠的第3代mASCs,通过流式细胞术测量 CD11b (0.6±0.2%),CD31 sup-(0%),CD45 sup-(0.03±0.06%),sca-1 < sup> + (80.3± 14.5%),CD29 +(99.9±0.1%)和CD44 +(99.8±0.2%)。表达方式 水平显示为表达相对标志物的群体的% 到相应的同种型对照染色(平均值±标准 偏差,n = 3)。表达范围可以在小鼠品系之间变化。 小鼠MSC分化成成骨细胞,脂肪细胞和 软骨细胞(Anderson等人,2013; Peister等人,2004)可以是 使用来自例如干细胞的商业分化试剂盒进行 技术( http://www.stemcell.com )和Lonza( http://www.lonza.com )。

  3. 扩散(注5)
    1. 准备mASCs(3-7代)用于增殖测定
      1. 如上所述收获细胞。
      2.  轻轻地,但彻底地重悬细胞沉淀在2毫升 Mesencult /汇合T75培养瓶中以获得5×细胞浓度 ?10 5个和1×10 6个细胞/ml。
      3. 稀释细胞悬液1:1在台盼蓝(20微升细胞悬浮液+ 20微升台盼蓝)。
      4. 计数。混合好,加入10μl到一个Neubauer计数室,并计数四个角的正方形(注6)。
      5. 准备10,000细胞/ml的工作溶液在完全Mesencult ?使用xCELLigence或6,600个细胞/ml测量细胞增殖 用于使用CellTiter-Blue试剂测量细胞增殖 将管保持在冰上(注7)。
    2. xCELLigence
      xCELLigence 系统可用于实时测量细胞增殖和使用 其中孔底部被覆盖的特定细胞培养板 电极(图1)。将细胞附着到电极增加 它们的阻抗被显示为小区索引的增加。的 细胞指数给出关于细胞数量,活力和形态的信息 ?。 xCELLigence RTCA站应放置在单元格中 设定在37℃,21%O 2,5%CO 2,90%RH的培养箱中。


      图1.组件 的xCELLigence系统。左侧面板显示了使用的16孔E盘 用于测量贴壁细胞的增殖。中间面板显示 DP RTCA站与其三个集成站为电子版。的 ?右侧面板给出了RTCA软件如何可视化单元格的示例 通过绘制细胞指数对时间的实时增殖。

      1. 在无菌罩中,在16孔中加入100μl/孔的完全Mesencult E盘,盖上盖子,放在xCELLigence RTCA站。 检查连接和电池索引读数,以确保 16孔E板正确安装。离开16孔E盘 ?xCELLigence RTCA站30-45分钟,然后测量背景 在没有细胞的情况下的阻抗
      2. 从中取出E板 xCELLigence RTCA站,并将其放置在无菌罩。涡旋管 ?含有10,000mASC/ml以重悬细胞并小心加入100μl ?μl的细胞悬液(在步骤C1e中制备)/孔,一式两份 小心不要触摸井的底部与提示。最后 体积/孔现??在应为200μl。
      3. 将mASCs留在细胞培养箱中的xCELLigence RTCA站中7天,每小时读取阻抗(注8)。
      4. 绘制细胞指数与培养物中的时间(参见下面的图2)。
    3. CellTiter-Blue
      这个 ?方法是基于刃天青到荧光的转换 代谢活性细胞产生试卤灵。荧光信号 ?获得的数量与培养物中的活细胞数量成比例。
      1. 加入150微升的细胞悬浮液/孔在平底96孔组织 培养板(每孔含约1,000个细胞) 一式三份。使1板/时间点。单独添加培养基 一式三份加入每个板中作为背景对照。放置 在设定为37℃,5%CO 2,21%O 2,90%RH的细胞培养箱中。
      2. 在第1,3和5-6天读板。加入30μlCellTiter-Blue 试剂/孔,轻轻敲打板的侧面并孵育该板 再保持4小时。
      3. 转移100微升从每个孔到黑色 96孔板并在荧光测定法中读取荧光(Glomax 多检测系统或等同物)在560 Ex/590/590 Em。
      4. 减去 ?来自样品荧光和图的平均背景荧光 荧光(增殖)对时间(参见下面的图2)

        图 ?2.代表性数据显示mASCs的增殖使用 xCELLigence系统(左图)和CellTiter-Blue试剂(右图) 面板)

笔记

  1. 用70%乙醇彻底喷雾处死小鼠的腹部,并使用医用注射器针头将动物,腹侧向上固定在聚苯乙烯支持物上。使用一套无菌剪刀/镊子仔细做一个中线切口,小心不要损伤腹膜。将皮肤与腹膜分开,用注射器针固定皮瓣。当获取腹股沟脂肪垫时,应注意去除在血管的Y形交叉处嵌入脂肪中的腹股沟淋巴结。使用一套新的无菌剪刀/镊子,收集腹股沟脂垫,并将其转移到含有10毫升HBSS的50毫升管,并保持在冰上。继续切开腹膜,注意不要损伤肠。收集附着在雄性和雌性小鼠的睾丸和卵巢上的生殖腺脂肪垫。当分离mASCs时,我们从两个位置汇集脂肪。然而,已经报道了MSC表型的贮库特异性差异,并且取决于您的科学目标,可能必须从一个位置或另一个位置分离mASC。
  2. 溶液可以是粘稠的,可能需要使用几个细胞过滤器
  3. 在以下步骤中的沉淀是松散的,并且在去除上清液时应该小心
  4. 我们将mASCs以10,000个细胞/cm 2的浓度接种,这允许我们每3-4天传代细胞。在这些培养条件下,我们已经能够使用xCELLigence系统或CellTiter-Blue试剂测量来自第3-7代的mASC的增殖。然而,应当注意,mASC的大小随着传代数的增加而增加,而它们的增殖能力降低。因此,当将它们的增殖能力与上述方法比较时,最好使用具有相同传代数的mASC细胞系。
  5. 我们提供测量mASC增殖的两个协议。 CellTiter-Blue试剂易于使用,在大多数实验室都可行。然而,一些研究已经显示,刃天青可以在长时间暴露时影响细胞活力,并且包含增加线粒体数量/活性的药物可以给出错误的结果。 xCELLigence系统需要购买RTCA站/软件和专用E盘的成本。然而,xCELLigence系统实现了无标记和非侵入性实时扩增测量。当在体外研究mASC增殖时,通常并行使用两种方法。要获得有关CellTiter-Blue试剂和xCELLigence系统的更多信息,请阅读制造商的说明。
  6. 我们优选计数平均20-50个细胞/角正方形,以获得可靠的细胞浓度。使用以下公式计算细胞浓度:(四个角正方形中的细胞总数/4)×2(稀释倍数)×10,000 =无。细胞/ml
  7. 我们发现添加1,000个mASC /孔(3,125个细胞/cm 2)允许在培养物达到融合之前监测mASC增殖5-7天。该细胞数目给出可重现的生长曲线,其包括根据图1的初始24-48h滞后期,接着是增殖期。
  8. 在实验过程中,我们通常不向培养板中加入新鲜培养基。然而,可以暂停实验,去除E板,添加另外100微升培养基/孔,并重新开始阻抗测量。

食谱

  1. I型胶原酶 将I型胶原酶(100mg)溶于10ml HBSS中至10mg/ml的浓度。
    将0.5ml的等分试样储存在-20℃下
  2. 完成MesenCult介质
    在4℃下融化MesenCult TM 干细胞刺激性补充剂(小鼠),稳定的谷氨酰胺,青霉素/链霉素,并混合:
    400ml MesenCult MSC基础培养基(小鼠)
    100 ml MesenCult干细胞刺激性补充剂(小鼠)
    5ml稳定的谷氨酰胺(终浓度:2mM) 5ml青霉素/链霉素(终浓度:100U/ml) 0.5 ml Mesenpure(可选)
    将完整的MesenCult存储在4°C,并在一个月内使用

致谢

这项工作由来自欧洲联盟的Fondo de Investigaciones Sanitarias ISCIII(西班牙)和Fondo Europeo de Desarrollo区域(FEDER)资助,通过研究拨款NoPI12/01390(PA),NoPI12/01097和ISCIII Red de Terapia Celular (授予NoP09-CTS-04532和PAIDI-Bio-326)和Consejeríade Salud(授予NoPI0001/2009)的产品(TerCel:RD12/0019/0006) deAndalucía和FEDER/Fondo de Cohesion Europeo(FSE)deAndalucía2007-2013 to FM P.A有一个Miguel Servet合同(CP09/00228),由来自欧盟的Fondo Europeo de Desarollo区域(FEDER)共同资助。调频。由FundaciónProgreso y Salud(Consejeríade Salud - Junta deAndalucía)资助。

参考文献

  1. Anderson,P.,Carrillo-Gálvez,A. B.,Garcia-Perez,A.,Cobo,M. andMartín,F。 CD105(内皮素) - 阴性鼠间质基质细胞定义了具有不同分化和免疫调节能力的新的多潜能亚群。 PLoS One 8(10):e76979。
  2. Carrillo-Gálvez,AB,Cobo,M.,Cuevas-Ocana,S.,Gutierrez-Guerrero,A.,Sanchez-Gilabert,A.,Bongarzone,P.,Garcia-Perez,A.,Munoz,P.,Benabdellah ,K.,Toscano,MG,Martín,F。和Anderson,P。(2015)。 间充质基质细胞在其表面表达GARP/LRRC32:对其生物学和免疫调节能力的影响。 a> Stem Cells 33(1):183-195。
  3. Peister,A.,Mellad,J.A.,Larson,B.L.,Hall,B.M.,Gibson,L.F.and Prockop,D.J。(2004)。 从不同种系的近交小鼠中分离的来自骨髓的成体干细胞(MSC)在表面表位,增殖速率和分化潜能。血液 103(5):1662-1668。
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How to cite this protocol: Anderson, P., Carrillo-Gálvez, A. B. and Martín, F. (2015). Isolation of Murine Adipose Tissue-derived Mesenchymal Stromal Cells (mASCs) and the Analysis of Their Proliferation in vitro. Bio-protocol 5(21): e1642. DOI: 10.21769/BioProtoc.1642; Full Text



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