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Sep 2018
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Cell Microencapsulation and Cryopreservation with Low Molecular Weight Hyaluronan and Dimethyl Sulfoxide
利用低分子量透明质酸和二甲基亚砜进行细胞微胶囊化和冷冻保存   

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Abstract

Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.

Keywords: Cryoprotectant agent (低温保护剂), Slow freezing (慢速冷冻), Hyaluronan (透明质酸), Dimethylsulfoxide (二甲基亚砜), Cell microencapsulation (细胞微胶囊化)

Background

Cell microencapsulation is extensively used to enclose cells allowing the exchange of nutrients between the environment and the core of the microcapsule containing the cells. In vivo, microcapsules protect the cells from the immune system while allowing the release of therapeutic molecules by the entrapped cells (Orive et al., 2014). Consequently, these advantages have promoted the development and employment of cell encapsulation in organ replacement, tissue engineering and regenerative medicine as drug and cell delivery therapies (Gurruchaga et al., 2015b). Nowadays the storage of microencapsulated cells is performed by slow freezing using a linear cooling rate (-0.3-1 °C/min) (Murua et al., 2009). Different cryoprotectant agents (CPAs) have been used for their cryopreservation being dimethylsulfoxide (DMSO) the most used with good results in a wide range of microencapsulated cells (Massie et al., 2011; Gurruchaga et al., 2015a; Gryshkov et al., 2015). However, DMSO has been related to several adverse reactions after the implantation of cryopreserved cell-based products (Shu et al., 2014; Ruiz-Delgado et al., 2009) such as cardiac arrhythmia, cardiac arrest, tonic-clonic seizure or diarrhea among others, being other alternative CPAs investigated to overcome the DMSO’s drawback (Mantri et al., 2015). In this context, low molecular weight hyaluronan (LMW-HA), a natural non-toxic CPA, has demonstrated cryoprotective effects in the cryopreservation of several cell types (Hutson et al., 2009; Ujihira et al., 2010; Iwama et al., 2014). Therefore, in this study, we have determined the cryoprotective effect of LMW-HA 5% for the cryopreservation of D1 mesenchymal stem cells (D1MSC) and murine C2C12 myoblast (C2C12) compared to DMSO 10% in each cell medium. LMW-HA 5% preserved the metabolic activity and cells viability similarly to DMSO containing solutions after thawing, showing the potential of LMW-HA as a CPAs. Although the use of LMW-HA as a CPA has been investigated in microencapsulated cells, it may represent an alternative CPA in the cryopreservation of other 3D cell-based products avoiding the use of DMSO.

Materials and Reagents

  1. Materials
    1. Pipette tips (Sharlab, catalog number: 00PC1000-1)
    2. 1.5 ml Eppendorf tubes (Sharlab, catalog number: 027200400P)
    3. T-175 flask (175 cm2) (Corning, catalog number: 431466)
    4. T-75 flask (75 cm2) (Corning, catalog number: 431464U)
    5. 15 ml centrifuges tubes (Corning, catalog number: 430791)
    6. 50 ml centrifuges tubes (Corning, catalog number: 430828)
    7. 96-well plates (Corning, catalog number: 353072)
    8. 24-well plates (Corning, catalog number: 3524)
    9. 5 ml polystyrene round bottom tubes (Corning, catalog number: 352052)
    10. Cryovials (Corning, catalog number: 430489) 
    11. 0.22 μm syringe filter NML plus (Minisart, catalog number: 17823)
    12. Stericup® (Merk Millipore, model: SCVPU02RE) 
    13. Cell strainer 40 and 100 μm (Corning, catalog number: 352360) 
    14. Luer 10 ml syringe (Braun, catalog number: 4606108V)

  2. Cells
    1. D1 mesenchymal stem cells (D1MSC) (ATCC, catalog number: CRL12424)
    2. Murine myoblast cells (C2C12) (ATCC, catalog number: CRL1772)

  3. Reagents
    1. Liquid nitrogen (Air liquid)
    2. Reverse osmosis water (Merck Milli Q system, catalog number: 7003/7005)
    3. Dubelcco’s Phosphate Buffered Saline (DPBS) (GIBCO, catalog number: D8537)
    4. DPBS containing Ca2+ and Mg2+ (Lonza, catalog number: 17-512F)
    5. Trypsin 0.25% (GIBCO, catalog number: 25200056)
    6. Mannitol (Sigma, catalog number: M4125)
    7. Calcium chloride (Sigma, catalog number: C4901)
    8. Ultra-pure low-viscosity and high glucuronic acid alginate (LVG) (Novamatrix, catalog number: BP-1410-19)
    9. Sodium citrate (tri-Sodium Citrate 2-hydrate) (Panreac Application, catalog number: 141655.1211)
    10. D1MSCs culture medium (ATCC, catalog number: DMEM 30-2002)
    11. C2C12 culture medium (Gibco, catalog number: DMEM 11960)
    12. Fetal bovine serum (FBS) (Gibco, catalog number: 42G9273K)
    13. Antibiotic/antimycotic (Gibco, catalog number: 15140-122)
    14. Dimethyl sulfoxide (DMSO) (ATCC, catalog number: A503039)
    15. Low molecular-weight hyaluronan 30-50 kD (LMW-HA) (Contipro, catalog number: 9067-32-7)
    16. Cell counting kit-8 (Sigma, catalog number: 96992-500TESTS-F)
    17. LIVE/DEADTM Viability/Cytotoxicity kit (488/570) (Invitrogen, catalog number: L3324)
    18. Annexin V-FITC Apoptosis Detection Kit (Sigma, catalog number: APOAF-50TST)
    19. D1MSC complete medium (see Recipes)
    20. C2C12 complete medium (see Recipes)
    21. DMSO 10% CPA solution (see Recipes)
    22. LMW-HA 5% CPA solution (see Recipes)
    23. 1% mannitol (see Recipes)
    24. 55 mM calcium chloride (see Recipes)
    25. 1% sodium citrate (see Recipes)
    26. 1.5% alginate LVG (NovaMatrix) (see Recipes)
    27. Calcein/ethidium staining solution (for microscopy) (see Recipes)
    28. Calcein/ethidium staining solution (for flow cytometry) (see Recipes)

Equipment

  1. Sterile spatula (Sharlbab, catalog number: 3100000BOC)
  2. Pipettes 2-20 μl, 20-200 μl, 100-1,000 μl (Eppendorf, catalog number: 4924000916)
  3. CoolCell® Cell Freezing Containers (Alcohol-free controlled-rate -1 °C/min cell freezing containers) (Corning Biocision, catalog number: BCS-405)
  4. -80 °C freezer (Thermo Fisher, catalog number: TSX40086A)
  5. TC20TM automated cell counter (Bio-Rad, catalog number: 145-0101)
  6. Water bath at 37 °C (Memmert, catalog number: WNB 45)
  7. Laminar flow hood (Burdinola, catalog number: AH-100)
  8. Centrifuge Mixtasel-BL (Selecta, catalog number: 7002575)
  9. CO2 incubator (Sanyo, catalog number: MCO-20 AIC)
  10. Fridge (Liebherr, catalog number: 12084121)
  11. Autoclave Autester ST (Selecta, catalog number: 4002517)
  12. Microscope (Nikon TMS microscope, catalog number: 310450)
  13. Liquid nitrogen tanks (Air liquid, catalog number: ARPEGE40-L-102)
  14. Vacuum pump (Millipore, catalog number: XF54 23050)
  15. FACS Calibur flow cytometerTM [Becton Dickinson (BD)]
  16. Infinite® M200 microplate reader (Tecan, model: Infinite® M200)
  17. Encapsulation process materials (Unless another company is indicated, all components are part of the system and purchased as a unit from Nisco encapsulation unit Var V1, catalog number: LIN-0203) (All materials are sterilized previously to cell microencapsulation):
    1. Electrostatic droplet generator
    2. Beaker (80 mm diameter, 40 mm height and 80 ml volume)
    3. Stirring
    4. Needle
    5. Needle holder
    6. Peristaltic pump
    7. Silicone tube
    8. Stainless steel stick
    9. Silicone tube
    10. Luer 10 ml syringe (Braun, catalog number: 4606108V)

Software

  1. Microsoft Office Excel (Microsoft)
  2. FlowJo® V10 (Flowjo, LLC)
  3. SPSS statistics 22 (IBM)
  4. Eclipse Net software, version 1.20.0 (Nikon)

Procedure

  1. Thawing and cell culture conditions
    1. When required, recover the D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) by rapidly thawing cryovials in a 37 °C water bath until no ice is observed (4-5 min). 
    2. Next, take cryovials into a laminar flow hood, transfer each cell suspension into a 50 ml conical centrifuge tube and dilute samples with 10 ml of complete culture medium.
    3. Spin the cell suspensions, remove the supernatant and resuspend each cell pellet with 20 ml of culture medium.
    4. Then, place each cell suspension separately into T-175 flasks and incubate them at 37 °C in a humidified 5% CO2/95% air atmosphere.
    5. Cultivate the cells in as many T-175 flasks as needed depending on the cell density that will be used for encapsulation, and the final volume of the batch that will be fabricated. Cells should be passaged when cell confluency is around 70% (every 2-3 days if 1 x 106 cells are seeded in a T-175 flask).

  2. Preparation of different cell suspension in alginate for microencapsulation
    1. Remove culture medium from the T-175 flasks, add 5 ml of PBS and remove again to take away remains of culture medium. 
    2. Add 5 ml of 0.25% trypsin and keep the flasks in the incubator at 37 °C for 2-5 min, after which, gently tap the side of the flask to dislodge the cells. 
    3. Remove cell suspension from the cell culture flask and transfer into a 50 ml conical flask.
    4. Filter the cell suspension through a 40 µm filter to remove cell aggregations and count the cells with an automatic cell counter or alternatively with a Neubauer Chamber.
    5. Collect the volume of cell suspension to get the final cell amount required for cell encapsulation.
    6. Spin the collected cell suspension (100 x g for 5 min) and discard the supernatant carefully.
    7. Resuspend cells by adding the needed volume of 1.5% alginate LVG solution (see Recipes) carefully to get 5 x 106 cells per ml of alginate density (for a 4 ml batch, 2 x 107 cells are needed). 
    8. Revolve cell suspension softly using a sterile spatula to homogenize it.
    Note: Each cell type is microencapsulated separately in this protocol.

  3. Cells microencapsulation
    1. Transfer the alginate cell suspension into a 10 ml sterile syringe avoiding the formation of bubbles. Place the syringe into a peristaltic pump (Figure 1A) (Video 1).

      Video 1. Electrostatic droplet generator assembly

    Note: All the microencapsulation procedure is performed in a biosafety cabinet. 
    1. Connect the syringe to the stainless steel needle tip (inner diameter 0.17 mm) with a silicone tube of 40 cm (Figure 1B).
    2. Fill the sterile beaker (80 mm diameter, 40 mm height and 80 ml volume) containing a small magnetic stirrer with 80 ml of the sterile calcium chloride gelling solution and place it under the needle (see Recipes). 
    3. Set up magnetic stirring to 200 rpm (this would keep the microcapsules separated during the gelling procedure).
    4. For a 380 µm diameter microcapsules, place the needle holder at 7 cm of height (Figure 1C), and be sure that the distance between the needle tip and the calcium chloride solution is of 2 cm.


      Figure 1. Cell microencapsulation in detail. A. Peristaltic pump set at 5.9 ml/h. B. Complete set-up of the Nisco® microcapsules generator. C. Detail of the set-up of the needle and its holder.

    5. Place the stainless steel stick inside the gelling bath and connect the electrode of the microcapsules generator.
    6. For 380 ± 10 µm diameter alginate microcapsules, set up the conditions of voltage (7 kV) and flow rate in the peristaltic pump (5.9 ml/h).
      Note: With the described conditions we obtain 380 ± 10 µm diameter alginate microcapsules that will slightly increase in diameter after coating. The bead diameter will decrease with smaller needle diameter and increasing voltage.
    7. Close the electrical safety cage (otherwise the system will not work). 
    8. Push the syringe manually until the alginate suspension reaches the tip of the nozzle (to take out the air from the silicone tube) and start the peristaltic pump at 5.9 ml/h speed. 
    9. Let the alginate solution to be pushed out into the beaker through the nozzle.
    10. When the syringe is empty, stop the flow of the pump and let the alginate microcapsules mixing with a magnetic stir at 200 rpm in the calcium chloride solution for at least 10 min for complete gelation. Collect microcapsules from calcium chloride solution by filtering the solution through a 100 µm strainer.
    11. Invert the 100 µm strainer and place it on the top of a conical tube.
    12. Next, collect microcapsules adding 10 ml of complete culture solution to the bottom of the strainer which is upside down. 
    13. Transfer the microcapsules into a T-75 flask with a pipette and add another 10 ml of complete medium.
    14. To confirm microcapsules integrity, shape, and homogeneity, observe them under an inverted microscope by phase contrast. Keep the flask in an incubator at 37 °C in a humidified 5% CO2/95% air atmosphere for further studies (Figure 2).
      Note: These microencapsulated cell types can be maintained in culture for at least 2 months.


      Figure 2. (A) C2C12 and (B) D1MSC cells in alginate microcapsules. Scale bar: 200 µm.

  4. Preparation and conditions for the cryopreservation process
    1. Precool the different CPA solutions to 4 °C (see Recipes). 
    2. Precool the CoolCell® container to 4 °C keeping in a fridge for more than 30 min. 
    3. Label 2 ml cryovials and let them on ice at 4 °C.

  5. Cryopreservation process
    1. Collect microcapsules from the T-75 flask and place them into a 1.5 ml Eppendorf tube. Let them sediment by gravity, and repeat this process until 200 µl of microcapsules are obtained. Next, remove the medium with a pipette (Figure 3).


      Figure 3. Sedimentation of microcapsules by gravity

    2. Add 1 ml of precooled CPA solution to the Eppendorf tubes (see Recipes).
      Note: CPA solutions can be prepared fresh, or they can be stored at most for 1 week at 4 °C.
    3. Transfer the microcapsules suspension to the 2 ml cryovials and keep them on ice 20 min (Figure 4).


      Figure 4. Chilling samples before cryopreservation

    4. Transfer the cryovials to the chilled Coolcell® containers.
    5. Introduce the containers in a -80 °C freezer for the slow controlled cooling of the samples, and keep them overnight.
    6. Next day, take out the Coolcell® container from the -80 °C freezer and transfer the cryovials to a liquid nitrogen tank for long-term storage.

  6. Microencapsulated cells recovery
    1. Carefully take out the cryovials from the liquid nitrogen tank. Transfer them as quick as possible to a 37 °C water bath for fast thawing.
    2. Move them softly in the water bath (3-5 min) trying to avoid the contact of the water with the cryovial lids to avoid contamination risk. On this regard, hold the cryovials from the tips or place them with a polyester holder that avoids tip contact with water.
    3. Transfer microcapsules from cryovials into 15 ml conical tubes, and dilute them adding 10 ml of culture medium at room temperature to the 15 ml tube drop by drop. Let the microcapsules to sediment by gravity and then remove the medium. Repeat the process at least twice.
    4. Add culture medium at 37 °C and incubate the microcapsules in a T-75 flask at 37 °C in a humidified 5% CO2/95% air atmosphere.

  7. Viability assessment of encapsulated cells under fluorescent microscopy
    1. Collect 20 µl of microcapsules from the T-75 flask and place it into a 1.5 ml Eppendorf tube. Let sediment by gravity and remove the medium. 
    2. Rinse the microcapsules three times (letting microcapsules settle by gravity) each with 1 ml of DPBS containing Ca2+ and Mg2+.
    3. Dilute rinsed microcapsules with 300 µl of calcein/ethidium staining solution for microscopy (see Recipes).
    4. Dispense this staining solution containing microcapsules into 3 wells of a 96-well plate (approximately 100 µl solution/well).
    5. Incubate at room temperature for 45 min protected from light. 
    6. Assess under a fluorescent microscope the viability of encapsulated cells (Figure 5). Calcein (excitation 495 nm, emission 515 nm for viable cells) and ethidium (excitation 495 nm, emission 635 nm). The fluorescence from these dyes may be observed separately; calcein with a standard fluorescein bandpass filter and ethidium with filters for propidium iodide or Texas Red® dye.


      Figure 5. Micrographs of calcein/ethidium stained cryopreserved microencapsulated cells 7 days in culture after thawing. Scale bars: 200 µm.

  8. Determination of the metabolic activity of microencapsulated cells
    1. Collect 50 µl of microcapsules from the T-75 flask and place it into a 1.5 ml Eppendorf tube and let it sediment by gravity, and next remove the medium. 
    2. Resuspend the encapsulated cells in 700 µl of complete culture medium.
    3. Dispense this solution containing the microcapsules into 7 wells of a 96-well plate (100 µl solution/well). Also, dispense 100 µl of just complete culture medium into 2 wells (negative control).
    4. Add 10 µl of solution for viability quantification (CCK-8) into each well.
    5. Incubate the 96 well-plate inside a wet chamber on an incubator for 4 h at 37 °C.
    6. After 4 h, transfer the supernatants into another 96-well plate.
    7. Measure the absorbance at 450 nm with reference wavelength at 650 nm using Infinite® M200 microplate reader or another microplate reader (Figure 6).


      Figure 6. Metabolic activity of cryopreserved microencapsulated (A) C2C12 and (B) D1MSC cells on Days 1 and 7 after thawing

  9. Viability quantification of microencapsulated cells by flow cytometry
    1. Collect 100 µl of microcapsule, corresponding to 5 x 105 cells, from the T-75 flask and place it into a 1.5 ml Eppendorf tube and let it sediment by gravity, remove next to the medium.
    2. Rinse twice the microcapsules with DPBS.
    3. Remove the supernatant and add 100 µl of 1% sodium citrate (see Recipes). Pipette up and down until microcapsules are dissolved.
    4. Filter the sample through a 40 µm cell strainer into different 15 ml conical tubes. 
    5. Centrifuge the tubes at 100 x g for 10 min and discard the supernatant to remove the rests of alginate. 
    6. Add the staining solution (1 ml for each sample) and incubate them for 20 min protected from light (see Recipes). Control samples are needed for cytometer calibrating (one without dyes, other with calcein and another one with ethidium). 
    7. Transfer samples to 5 ml round bottom tubes to quantify cell viability in the FACS Calibur flow cytometerTM (Figure 7).


      Figure 7. Quantification of dead cells in cryopreserved microencapsulated (A) C2C12 and (B) D1MSC cells on Days 1 and 7 after thawing. Values represent mean ± SD. **: P ˂ 0.01 and ***: P ˂ 0.001 compared to HA 5%.

Data analysis

All quantifications are conducted in triplicates, with at least three independent experiments. The mean and standard deviation are calculated by functions ‘AVERAGE’ and ‘STDEV’ respectively in Microsoft Office Excel, and the statistical analysis are performed with SPSS software. Cytometer data analysis is performed with FlowJo® V10 (Flowjo, LLC). Microscope micrographs are analyzed with Eclipse Net software, version 1.20.0.
  The metabolic activity values increased as expected from Day 1 to Day 7 in each cryopreserved microencapsulated cell type independently of the CPA used due to cell proliferation within microcapsules (Figure 6). Moreover, dead cells percentages increased from Day 1 to Day 7 in each cryopreserved microencapsulated cell type independently of the CPA used (Figure 7). The increase in dead cell percentage can be explained with the cryopreservation-induced onset cell death phenomena, as cell death values are increased after 24 h of thawing.

Recipes

  1. D1MSC complete medium
    From 500 ml DMEM (ATCC) bottle remove 10% of volume and add 1% volume of antibiotic/antimycotic solution, and add 10% volume of the FBS
  2. C2C12 complete medium
    From 500 ml DMEM (Gibco) bottle remove 10% of volume and add 1% volume of antibiotic/antimycotic solution, and 10% volume of the FBS
  3. DMSO 10% CPA solution
    Add 1 ml of DMSO in 9 ml of each complete cell culture medium and store it at 4 °C
  4. LMW-HA 5% CPA solution (20 ml)
    1. Resuspend 200 mg LMW-HA powder in 10 ml of complete medium and stir until is dissolved under aseptic conditions
    2. When dissolved add 10 ml of complete medium and store it at 4 °C
  5. 1% mannitol
    1. Dissolve 1 g of mannitol into 100 ml of distilled water or scale up to the required volume
    2. Stir until mannitol gets dissolved and filter through a Millipore 0.22 µm filter Stericup®
  6. 55 mM calcium chloride (500 ml)
    1. Dissolve 2.44 g of calcium chloride (96% purity) into 500 ml of 1% mannitol solution
    2. Scale up the amounts of calcium chloride to higher volumes
    3. Stir until it dissolves, and filter through a Millipore 0.22 µm filter Stericup®
  7. 1% sodium citrate
    1. Dissolve 1 g of sodium citrate into 100 ml of 1% mannitol or scale up to the required volume
    2. Stir until it gets dissolved and filter through a Millipore 0.22 µm filter Stericup®
  8. 1.5% alginate LVG (NovaMatrix)
    1. Dissolve 1.5 g alginate into 100 ml of 1% mannitol and stir
    2. When alginate solution is transparent, filter through a 0.20 µm syringe filter
    Note: For higher volumes of alginate solution, scale up as required. Lower or higher alginate concentrations will require you to modify the weight of alginate dissolved into 1% mannitol. We recommend filtering on steps of 2 ml since higher alginate volumes require strength to get filtered due to its high viscosity.
  9. Calcein/ethidium staining solution (for microscopy)
    Dilute 5 µl of ethidium and 5 µl of calcein provided in the LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Life-Technologies) into 10 ml of DPBS containing Ca2+ and Mg2+
  10. Calcein/ethidium staining solution (for flow cytometry)
    1. Prepare a calcein stock of 100 µM solution diluting 4 µl of calcein provided in the LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Life-Technologies) in 156 µl of DMSO 
    2. For control with only calcein, add 2 µl of calcein stock in 1 ml of cell culture medium 
    3. For control with only ethidium, add 4 µl of ethidium provided in the LIVE/DEAD® Viability/Cytotoxicity Kit in 1 ml of cell culture medium 
    4. For each sample, add 2 µl of calcein stock and add 4 µl of ethidium in 1 ml of cell culture medium
    Note: Use cell culture medium of each cell type without serum and Antibiotic/antimycotic.

Acknowledgments

This protocol was adapted from the previously published study (Murua et al., 2009) and (Gurruchaga et al., 2015a) Author thanks the University of the Basque Country (UPV/EHU) for granted fellowship. Authors also wish to thank the intellectual and technical assistance from the ICTS “NANBIOSIS”, more specifically by the Drug Formulation Unit (U10) of the CIBER-BBN at the UPV/EHU.

Competing interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

References

  1. Gryshkov, O., Hofmann, N., Lauterboeck, L., Pogozhykh, D., Mueller, T. and Glasmacher, B. (2015). Multipotent stromal cells derived from common marmoset Callithrix jacchus within alginate 3D environment: Effect of cryopreservation procedures. Cryobiology 71(1): 103-111.
  2. Gurruchaga, H., Ciriza, J., Saenz Del Burgo, L., Rodriguez-Madoz, J. R., Santos, E., Prosper, F., Hernandez, R. M., Orive, G. and Pedraz, J. L. (2015a). Cryopreservation of microencapsulated murine mesenchymal stem cells genetically engineered to secrete erythropoietin. Int J Pharm 485(1-2): 15-24.
  3. Gurruchaga, H., Saenz del Burgo, L., Ciriza, J., Orive, G., Hernandez, R. M. and Pedraz, J. L. (2015b). Advances in cell encapsulation technology and its application in drug delivery. Expert Opin Drug Deliv 12(8): 1251-1267.
  4. Hutson, E. L., Coleman, C.M., Freestone, S. F., Huckle, J., Murphy, M. and Barry, F. (2009). Hyaluronic acid as a cryopreservant of mesenchymal stem cells. 55th Annual Meeting of the Orthopaedic Research Society. 
  5. Iwama, A., Yamada, C., Uchida, K. and Ujihira, M. (2014). Pre-incubation with hyaluronan reduces cellular damage after cryopreservation in densely cultured cell monolayers. Biomed Mater Eng 24(2): 1497-1506.
  6. Mantri, S., Kanungo, S. and Mohapatra, P. C. (2015). Cryoprotective effect of disaccharides on cord blood stem cells with minimal use of DMSO. Indian J Hematol Blood Transfus 31(2): 206-212.
  7. Massie, I., Selden, C., Morris, J., Hodgson, H. and Fuller, B. (2011). Cryopreservation of encapsulated liver spheroids using a cryogen-free cooler: high functional recovery using a multi-step cooling profile. Cryo Letters 32(2): 158-165.
  8. Murua, A., Orive, G., Hernandez, R. M. and Pedraz, J. L. (2009). Cryopreservation based on freezing protocols for the long-term storage of microencapsulated myoblasts. Biomaterials 30(20): 3495-3501.
  9. Orive, G., Santos, E., Pedraz, J. L. and Hernandez, R. M. (2014). Application of cell encapsulation for controlled delivery of biological therapeutics. Adv Drug Deliv Rev 67-68: 3-14.
  10. Ruiz-Delgado GJ, Mancías-Guerra C, Tamez-Gómez EL, Rodríguez-Romo LN, López-Otero A, Hernández-Arizpe A, Gómez-Almaguer D, Ruiz-Argüelles GJ. (2009). Dimethyl sulfoxide-induced toxicity in cord blood stem cell transplantation: report of three cases and review of the literature. Acta Haematol 122(1):1-5.
  11. Shu, Z., Heimfeld, S. and Gao, D. (2014). Hematopoietic SCT with cryopreserved grafts: adverse reactions after transplantation and cryoprotectant removal before infusion. Bone Marrow Transplant 49(4): 469-476. 
  12. Ujihira, M., Iwama, A., Aoki, M., Aoki, K., Omaki, S., Goto, E. and Mabuchi, K. (2010). Cryoprotective effect of low-molecular-weight hyaluronan on human dermal fibroblast monolayers. Cryo Letters 31(2): 101-111.

简介

低温保存通常用于使用超低温储存细胞,组织,器官或基于3D细胞的产品,其涉及浸入液氮中以进行长期保存。通常通过用二甲基亚砜(DMSO)作为冷冻保护剂(CPA)缓慢冷冻来进行几种微囊化细胞的冷冻保存。在这项研究中,我们用5%的天然无毒低分子量透明质酸(LMW-HA)和DMSO 10%溶液冷冻保存几个微囊化细胞,评估解冻后的细胞活力和代谢活性。与DMSO溶液相比,微囊化D1间充质干细胞(D1MSC)和小鼠成肌细胞(C2C12)与LMW-HA 5%的低温保存在解冻后呈现相似的结果,显示低分子量透明质酸作为天然无毒CPA可以用于在植入冷冻保存的基于细胞的产品后防止DMSO相关的副作用。
【背景】细胞微囊化广泛用于包封细胞,允许环境与含有细胞的微胶囊核心之间的营养物交换。 体内,微胶囊保护细胞免受免疫系统的影响,同时允许被诱捕的细胞释放治疗分子(Orive et al。,2014)。因此,这些优点促进了细胞包封在器官替代,组织工程和再生医学中作为药物和细胞递送疗法的开发和应用(Gurruchaga 等人,,2015b)。目前,微囊化细胞的储存通过使用线性冷却速率(-0.3-1℃/ min)缓慢冷冻来进行(Murua 等,,2009))。不同的冷冻保护剂(CPA)已用于冷冻保存,二甲基亚砜(DMSO)使用最多,在广泛的微囊化细胞中具有良好的效果(Massie et al。,2011; Gurruchaga et al。,2015a; Gryshkov et al。,2015)。然而,DMSO与植入冷冻保存的细胞产品后的几种不良反应有关(Shu et al。,2014; Ruiz-Delgado et al。,2009)例如心律失常,心脏骤停,强直 - 阵挛性癫痫或腹泻等,是其他替代CPA,以克服DMSO的缺点(Mantri et al。,2015)。在这种情况下,低分子量透明质酸(LMW-HA),一种天然的无毒CPA,已经在几种细胞类型的冷冻保存中显示出低温保护作用(Hutson et al。,2009; Ujihira et al。,2010; Iwama et al。,2014)。因此,在本研究中,我们已确定LMW-HA 5%对低温保存D1间充质干细胞(D1MSC)和鼠C2C12成肌细胞(C2C12)的冷冻保护作用与每种细胞培养基中DMSO 10%相比。 LMW-HA 5%在解冻后与含有DMSO的溶液类似地保留了代谢活性和细胞活力,显示了LMW-HA作为CPA的潜力。尽管已经在微囊化细胞中研究了LMW-HA作为CPA的用途,但它可以代表在其他基于3D细胞的产品的冷冻保存中避免使用DMSO的替代CPA。

关键字:低温保护剂, 慢速冷冻, 透明质酸, 二甲基亚砜, 细胞微胶囊化

材料和试剂

  1. 材料
    1. 移液器吸头(Sharlab,目录号:00PC1000-1)
    2. 1.5毫升Eppendorf管(Sharlab,目录号:027200400P)
    3. T-175烧瓶(175 cm 2 )(Corning,目录号:431466)
    4. T-75烧瓶(75 cm 2 )(Corning,目录号:431464U)
    5. 15毫升离心管(Corning,目录号:430791)
    6. 50毫升离心管(Corning,目录号:430828)
    7. 96孔板(康宁,目录号:353072)
    8. 24孔板(康宁,目录号:3524)
    9. 5毫升聚苯乙烯圆底管(康宁,目录号:352052)
    10. Cryovials(康宁,目录号:430489) 
    11. 0.22μm注射器过滤器NML plus(Minisart,目录号:17823)
    12. Stericup ®(Merk Millipore,型号:SCVPU02RE) 
    13. 细胞过滤器40和100μm(康宁,目录号:352360) 
    14. Luer 10 ml注射器(Braun,目录号:4606108V)

  2. 细胞
    1. D1间充质干细胞(D1MSC)(ATCC,目录号:CRL12424)
    2. 小鼠成肌细胞(C2C12)(ATCC,目录号:CRL1772)

  3. 试剂
    1. 液氮(空气液体)
    2. 反渗透水(Merck Milli Q系统,目录号:7003/7005)
    3. Dubelcco的磷酸盐缓冲盐水(DPBS)(GIBCO,目录号:D8537)
    4. 含有Ca 2 + 和Mg 2 + 的DPBS(Lonza,目录号:17-512F)
    5. 胰蛋白酶0.25%(GIBCO,目录号:25200056)
    6. 甘露醇(西格玛,目录号:M4125)
    7. 氯化钙(Sigma,目录号:C4901)
    8. 超纯低粘度和高葡萄糖醛酸海藻酸盐(LVG)(Novamatrix,目录号:BP-1410-19)
    9. 柠檬酸钠(柠檬酸三钠二水合物)(Panreac申请,目录号:141655.1211)
    10. D1MSCs培养基(ATCC,目录号:DMEM 30-2002)
    11. C2C12培养基(Gibco,目录号:DMEM 11960)
    12. 胎牛血清(FBS)(Gibco,目录号:42G9273K)
    13. 抗生素/抗真菌药(Gibco,目录号:15140-122)
    14. 二甲基亚砜(DMSO)(ATCC,目录号:A503039)
    15. 低分子量透明质酸30-50 kD(LMW-HA)(Contipro,目录号:9067-32-7)
    16. 细胞计数试剂盒-8(Sigma,目录号:96992-500TESTS-F)
    17. LIVE / DEAD TM 活力/细胞毒性试剂盒(488/570)(Invitrogen,目录号:L3324)
    18. Annexin V-FITC凋亡检测试剂盒(Sigma,目录号:APOAF-50TST)
    19. D1MSC完全培养基(见食谱)
    20. C2C12完全培养基(见食谱)
    21. DMSO 10%CPA溶液(参见食谱)
    22. LMW-HA 5%CPA解决方案(参见食谱)
    23. 1%甘露醇(见食谱)
    24. 55 mM氯化钙(见食谱)
    25. 1%柠檬酸钠(见食谱)
    26. 1.5%藻酸盐LVG(NovaMatrix)(见食谱)
    27. 钙黄绿素/乙锭染色溶液(用于显微镜检查)(见食谱)
    28. 钙黄绿素/乙锭染色溶液(用于流式细胞仪)(见食谱)

设备

  1. 无菌抹刀(Sharlbab,目录号:3100000BOC)
  2. 移液器2-20μl,20-200μl,100-1,000μl(Eppendorf,目录号:4924000916)
  3. CoolCell ®细胞冷冻容器(无酒精控制率-1°C / min细胞冷冻容器)(Corning Biocision,目录号:BCS-405)
  4. -80°C冰箱(Thermo Fisher,目录号:TSX40086A)
  5. TC20 TM 自动细胞计数器(Bio-Rad,目录号:145-0101)
  6. 37°C水浴(Memmert,目录号:WNB 45)
  7. 层流罩(Burdinola,目录号:AH-100)
  8. 离心机Mixtasel-BL(Selecta,目录号:7002575)
  9. CO 2 培养箱(三洋,目录号:MCO-20 AIC)
  10. 冰箱(利勃海尔,目录号:12084121)
  11. Autoclave Autester ST(Selecta,目录号:4002517)
  12. 显微镜(尼康TMS显微镜,目录号:310450)
  13. 液氮罐(空气液体,目录号:ARPEGE40-L-102)
  14. 真空泵(Millipore,目录号:XF54 23050)
  15. FACS Calibur流式细胞仪 TM [Becton Dickinson(BD)]
  16. 无限® M200酶标仪(Tecan,型号:Infinite ® M200)
  17. 封装工艺材料(除非指明其他公司,否则所有组件均为系统的一部分,并作为一个单元从Nisco封装单元Var V1购买,目录号:LIN-0203)(所有材料先前已对细胞微胶囊进行灭菌):
    1. 静电液滴发生器
    2. 烧杯(直径80毫米,高40毫米,体积80毫升)
    3. 搅拌
    4. 针座
    5. 蠕动泵
    6. 硅胶管
    7. 不锈钢棒
    8. 硅胶管
    9. Luer 10 ml注射器(Braun,目录号:4606108V)

软件

  1. Microsoft Office Excel(微软)
  2. FlowJo ® V10(Flowjo,LLC)
  3. SPSS statistics 22(IBM)
  4. Eclipse Net软件,版本1.20.0(尼康)

程序

  1. 解冻和细胞培养条件
    1. 需要时,通过在37°C水浴中快速解冻冷冻管直至没有观察到冰(4-5分钟),恢复D1间充质干细胞(D1MSC)和鼠成肌细胞(C2C12)。 
    2. 接下来,将冷冻管置于层流罩中,将每个细胞悬浮液转移到50ml锥形离心管中,并用10ml完全培养基稀释样品。
    3. 旋转细胞悬浮液,除去上清液,用20ml培养基重悬每个细胞沉淀。
    4. 然后,将每个细胞悬浮液分别置于T-175烧瓶中,并在37℃,湿润的5%CO 2 / 95%空气气氛中孵育。
    5. 根据将用于包封的细胞密度以及将要制造的批次的最终体积,根据需要在尽可能多的T-175培养瓶中培养细胞。当细胞融合率约为70%时,细胞应传代(如果1×10 6 细胞接种于T-175培养瓶中,则每2-3天传代一次)。

  2. 用于微胶囊化的海藻酸盐中不同细胞悬浮液的制备
    1. 从T-175烧瓶中取出培养基,加入5 ml PBS,再次取出,取出培养基残留物。 
    2. 加入5毫升0.25%胰蛋白酶,将烧瓶保持在37℃的培养箱中2-5分钟,然后轻轻敲打烧瓶侧面以去除细胞。 
    3. 从细胞培养瓶中取出细胞悬浮液,转移到50ml锥形瓶中。
    4. 通过40μm过滤器过滤细胞悬浮液以去除细胞聚集并用自动细胞计数器或者用Neubauer Chamber计数细胞。
    5. 收集细胞悬浮液的体积以获得细胞包封所需的最终细胞量。
    6. 旋转收集的细胞悬浮液(100 x g 5分钟)并小心弃去上清液。
    7. 通过小心地添加所需体积的1.5%藻酸盐LVG溶液(参见配方)重悬细胞,以获得每ml藻酸盐密度5×10 6个细胞(对于4ml批次,2×10 需要7个细胞。) 
    8. 使用无菌刮刀轻柔地旋转细胞悬浮液以使其均匀化。
    注意:每种细胞类型都在此协议中单独进行微囊化。

  3. 细胞微囊化
    1. 将藻酸盐细胞悬浮液转移到10ml无菌注射器中,避免形成气泡。将注射器放入蠕动泵(图1A)(视频1)。
      视频1.静电液滴发生器组件

    注意:所有的微胶囊化程序都在生物安全柜中进行。
    1. 将注射器连接到不锈钢针尖(内径0.17 mm),硅胶管为40 cm(图1B)。
    2. 将含有小磁力搅拌器的无菌烧杯(80毫米直径,40毫米高度和80毫升体积)装入80毫升无菌氯化钙胶凝溶液中,并将其放在针头下(参见食谱)。 
    3. 将磁力搅拌设定为200rpm(这将在胶凝过程中保持微胶囊分离)。
    4. 对于380微米直径的微胶囊,将针座置于7厘米高(图1C),并确保针尖与氯化钙溶液之间的距离为2厘米。


      图1.细胞微囊化详细信息。 A.蠕动泵设定为5.9 ml / h。 B.完成Nisco ®微胶囊发生器的设置。 C.针头及其固定器的设置细节。

    5. 将不锈钢棒放在胶凝浴内,并连接微胶囊发生器的电极。
    6. 对于380±10μm直径的藻酸盐微胶囊,在蠕动泵(5.9ml / h)中设定电压(7kV)和流速的条件。
      注意:在所描述的条件下,我们获得380±10μm直径的藻酸盐微胶囊,其在涂布后将直径略微增加。随着针头直径的减小和电压的增加,珠子直径会减小。
    7. 关闭电气安全笼(否则系统将无法工作)。 
    8. 手动按下注射器,直到藻酸盐悬浮液到达喷嘴尖端(从硅胶管中取出空气)并以5.9毫升/小时的速度启动蠕动泵。 
    9. 将藻酸盐溶液通过喷嘴推入烧杯中。
    10. 当注射器是空的时,停止泵的流动并让藻酸盐微胶囊在氯化钙溶液中以200rpm的磁力搅拌混合至少10分钟以完全凝胶化。通过100μm过滤器过滤溶液,从氯化钙溶液中收集微胶囊。
    11. 将100μm过滤器倒置并将其放在锥形管的顶部。
    12. 接下来,收集微胶囊,将10毫升完全培养液加入过滤器底部,倒置。 
    13. 用移液管将微胶囊转移到T-75烧瓶中,再加入10ml完全培养基。
    14. 为了确认微胶囊的完整性,形状和均匀性,在倒置显微镜下通过相差来观察它们。将烧瓶置于37℃,湿润的5%CO 2 / 95%空气气氛的培养箱中进行进一步研究(图2)。
      注意:这些微囊化细胞类型可在培养基中维持至少2个月。


      图2.(A)藻酸盐微胶囊中的C2C12和(B)D1MSC细胞。比例尺:200μm。

  4. 冷冻保存过程的准备和条件
    1. 将不同的CPA溶液预冷至4°C(参见食谱)。 
    2. 将CoolCell ®容器预冷至4°C,在冰箱中保存30分钟以上。 
    3. 标记2 ml冷冻管,置于4°C冰上。

  5. 冷冻保存过程
    1. 从T-75烧瓶中收集微胶囊并将其置于1.5ml Eppendorf管中。让它们通过重力沉淀,并重复该过程直至获得200μl微胶囊。接下来,用移液管移除培养基(图3)。


      图3.重力沉积微胶囊

    2. 在Eppendorf管中加入1 ml预冷的CPA溶液(参见食谱)。
      注意:CPA溶液可以新鲜制备,也可以在4°C下最多储存1周。
    3. 将微胶囊悬浮液转移至2 ml冷冻管中,并将其保持在冰上20分钟(图4)。


      图4.低温保存前冷却样品

    4. 将冷冻管转移至冷却的Coolcell ®容器中。
    5. 将容器放入-80°C冰箱中,对样品进行缓慢控制冷却,并将其保持过夜。
    6. 第二天,从-80°C冰箱中取出Coolcell ®容器,将冷冻瓶转移到液氮罐中进行长期储存。

  6. 微囊化细胞恢复
    1. 小心地从液氮罐中取出冷冻瓶。尽快将它们转移到37°C水浴中快速解冻。
    2. 将它们轻轻地在水浴中移动(3-5分钟),试图避免水与冷冻盒盖接触,以避免污染风险。在这方面,从尖端握住冷冻管或将其放置在聚酯支架上,避免尖端与水接触。
    3. 将微胶囊从冷冻管转移到15ml锥形管中,并在室温下将10ml培养基稀释至15ml管中逐滴稀释。让微胶囊通过重力沉淀,然后除去培养基。重复此过程至少两次。
    4. 在37℃下加入培养基,并将微胶囊在T-75烧瓶中于37℃在潮湿的5%CO 2 / 95%空气气氛中温育。

  7. 荧光显微镜下包囊细胞的活力评估
    1. 从T-75烧瓶中收集20μl微胶囊,并将其置于1.5ml Eppendorf管中。让沉淀物通过重力去除介质。 
    2. 将微胶囊冲洗三次(让微胶囊通过重力沉降)各自用1ml含有Ca 2+ +上的DPBS和Mg 2 + 。
    3. 用300μl钙黄绿素/乙锭染色溶液稀释漂洗的微胶囊用于显微镜检查(参见食谱)。
    4. 将含有微胶囊的染色溶液分配到96孔板的3个孔中(约100μl溶液/孔)。
    5. 在室温下孵育45分钟,避光。 
    6. 在荧光显微镜下评估包封细胞的活力(图5)。钙黄绿素(激发495nm,活细胞发射515nm)和乙锭(激发495nm,发射635nm)。可以分别观察这些染料的荧光;钙黄绿素与标准荧光素带通滤光片和乙锭与过滤器的碘化丙锭或德克萨斯红®染料。


      图5.解冻后培养7天时钙黄绿素/乙锭染色的冷冻保存的微囊化细胞的显微照片。比例尺:200μm。

  8. 微囊化细胞代谢活性的测定
    1. 从T-75烧瓶中收集50μl微胶囊,将其放入1.5 ml Eppendorf管中,让其在重力作用下沉淀,然后取出培养基。 
    2. 将包封的细胞重悬于700μl完全培养基中。
    3. 将含有微胶囊的该溶液分配到96孔板的7个孔中(100μl溶液/孔)。另外,将100μl刚刚完全培养基分配到2个孔中(阴性对照)。
    4. 向每个孔中加入10μl用于活力定量的溶液(CCK-8)。
    5. 将96孔板在培养箱中的湿室内孵育4小时,温度为37℃。
    6. 4小时后,将上清液转移到另一个96孔板中。
    7. 使用Infinite ® M200酶标仪或另一种酶标仪(图6),在650 nm处测量吸光度,参比波长为650 nm。


      图6.解冻后第1天和第7天冷冻保存的微囊化(A)C2C12和(B)D1MSC细胞的代谢活性

  9. 流式细胞术对微囊化细胞的活力定量分析
    1. 从T-75烧瓶中收集100μl相当于5×10 5个细胞的微胶囊,并将其置于1.5ml Eppendorf管中,让其在重力作用下沉淀,在培养基旁边除去。
    2. 用DPBS冲洗两次微胶囊。
    3. 除去上清液,加入100μl1%柠檬酸钠(参见食谱)。上下移液直至微胶囊溶解。
    4. 将样品通过40μm细胞过滤器过滤到不同的15 ml锥形管中。 
    5. 将管在100μL离心管中离心10分钟,弃去上清液以除去藻酸盐的残留物。 
    6. 加入染色溶液(每个样品1毫升),孵育20分钟避光(见食谱)。细胞计数器校准需要对照样品(一种不含染料,另一种含有钙黄绿素,另一种含有乙锭)。 
    7. 将样品转移至5 ml圆底管,以在FACS Calibur流式细胞仪 TM 中定量细胞活力(图7)。


      图7.解冻后第1天和第7天冷冻保存的微囊化(A)C2C12和(B)D1MSC细胞中死细胞的定量。值代表平均值±SD。 **: P ˂0.01和***: P ˂0.001与HA 5%相比。

数据分析

所有定量均一式三份进行,至少进行三次独立实验。平均值和标准差分别由Microsoft Office Excel中的函数“AVERAGE”和“STDEV”计算,统计分析使用SPSS软件进行。使用FlowJo ® V10(Flowjo,LLC)进行细胞分析仪数据分析。使用Eclipse Net软件版本1.20.0分析显微镜显微照片。
 在每个冷冻保存的微囊化细胞类型中,从第1天至第7天预期的代谢活性值增加,与由于微囊内的细胞增殖而使用的CPA无关(图6)。此外,在每个冷冻保存的微囊化细胞类型中死细胞百分比从第1天到第7天增加,与所使用的CPA无关(图7)。死细胞百分比的增加可以用冷冻保存诱导的起始细胞死亡现象来解释,因为在解冻24小时后细胞死亡值增加。

食谱

  1. D1MSC完整媒体
    从500ml DMEM(ATCC)瓶中取出10%体积并加入1%体积的抗生素/抗真菌溶液,并加入10%体积的FBS
  2. C2C12完全中等
    从500ml DMEM(Gibco)瓶中取出10%体积并加入1%体积的抗生素/抗真菌溶液和10%体积的FBS
  3. DMSO 10%CPA溶液
    在9ml每种完全细胞培养基中加入1ml DMSO,并将其储存在4℃
  4. LMW-HA 5%CPA溶液(20 ml)
    1. 将200mg LMW-HA粉末重悬于10ml完全培养基中并搅拌直至在无菌条件下溶解
    2. 溶解后,加入10毫升完全培养基,并保存在4°C
  5. 1%甘露醇
    1. 将1克甘露醇溶于100毫升蒸馏水中或按比例放大至所需体积
    2. 搅拌直至甘露醇溶解并通过Millipore0.22μm过滤器过滤Stericup ®
  6. 55 mM氯化钙(500 ml)
    1. 将2.44g氯化钙(96%纯度)溶解在500ml 1%甘露醇溶液中
    2. 将氯化钙的量增加到更高的体积
    3. 搅拌直至溶解,并通过Millipore0.22μm过滤器Stericup ®过滤
  7. 1%柠檬酸钠
    1. 将1克柠檬酸钠溶于100毫升1%甘露醇中或按比例放大至所需体积
    2. 搅拌直至其溶解并通过Millipore0.22μm过滤器Stericup ®过滤
  8. 1.5%藻酸盐LVG(NovaMatrix)
    1. 将1.5g藻酸盐溶解于100ml 1%甘露醇中并搅拌
    2. 当藻酸盐溶液透明时,通过0.20μm注射器过滤器过滤
    注意:对于更高体积的藻酸盐溶液,请根据需要进行放大。较低或较高的藻酸盐浓度将要求您修改溶解在1%甘露醇中的藻酸盐的重量。我们建议过滤2毫升的步骤,因为较高的藻酸盐体积需要强度才能过滤,因为它的粘度很高。
  9. 钙黄绿素/乙锭染色液(用于显微镜检查)
    将用于哺乳动物细胞的LIVE / DEAD ®活力/细胞毒性试剂盒(Life-Technologies)中提供的5μl乙锭和5μl钙黄绿素稀释到10 ml含有Ca 2 +的DPBS中和Mg 2 +
  10. 钙黄绿素/乙锭染色液(用于流式细胞仪)
    1. 制备100μM溶液的钙黄绿素储备液,稀释用于哺乳动物细胞(Life-Technologies)的LIVE / DEAD ®生存力/细胞毒性试剂盒中提供的4μl钙黄绿素,156μlDMSO 
    2. 对于仅使用钙黄绿素的对照,在1ml细胞培养基中加入2μl钙黄绿素原液 
    3. 对于仅含有乙锭的对照,在LIVE / DEAD ®活力/细胞毒性试剂盒中加入1μl细胞培养基中提供的4μl乙锭 
    4. 对于每个样品,加入2μl钙黄绿素原液,并在1ml细胞培养基中加入4μl乙锭
    注意:使用不含血清和抗生素/抗真菌剂的每种细胞类型的细胞培养基。

致谢

该协议改编自先前发表的研究(Murua et al。,2009)和(Gurruchaga et al。,2015a)作者感谢巴斯克大学(UPV) / EHU)理所当然的团契。作者还要感谢ICTS“NANBIOSIS”的知识和技术援助,更具体地说是感谢UPV / EHU的CIBER-BBN的药物制剂单元(U10)。

利益争夺

作者与任何与该手稿中讨论的主题或材料有财务利益或财务冲突的组织或实体没有相关的从属关系或财务参与。这包括就业,咨询,酬金,股票所有权或期权,专家证词,已获得或正在申请的赠款或专利,或特许权使用费。

参考

  1. Gryshkov,O.,Hofmann,N.,Lauterboeck,L.,Pogozhykh,D.,Mueller,T。和Glasmacher,B。(2015)。 藻酸盐3D环境中来自普通mar猴 Callithrix jacchus 的多能基质细胞:冷冻保存程序的效果。 低温生物学 71(1):103-111。
  2. Gurruchaga,H.,Ciriza,J.,Saenz Del Burgo,L.,Rodriguez-Madoz,J.R.,Santos,E.,Prosper,F.,Hernandez,R.M.,Orive,G。和Pedraz,J.L。(2015a)。 冷冻保存微胶囊化的鼠间充质干细胞,通过基因工程分泌促红细胞生成素。 Int J Pharm 485(1-2):15-24。
  3. Gurruchaga,H.,Saenz del Burgo,L.,Ciriza,J.,Orive,G.,Hernandez,R。M. and Pedraz,J.L。(2015b)。 细胞包封技术及其在药物输送中的应用进展。 Expert Opin Drug Deliv 12(8):1251-1267。
  4. Hutson,E.L.,Coleman,C.M.,Freestone,S.F.,Huckle,J.,Murphy,M。和Barry,F。(2009)。 透明质酸作为间充质干细胞的冷冻保存剂。第55届骨科研究年会社会 
  5. Iwama,A.,Yamada,C.,Uchida,K。和Ujihira,M。(2014)。 与透明质酸预孵育可减少细胞密集培养细胞单层中冷冻保存后的细胞损伤。 em> Biomed Mater Eng 24(2):1497-1506。
  6. Mantri,S.,Kanungo,S。和Mohapatra,P。C.(2015)。 使用极少使用DMSO的二糖对脐带血干细胞的低温保护作用。 Indian J Hematol Blood Transfus 31(2):206-212。
  7. Massie,I.,Selden,C.,Morris,J.,Hodgson,H。和Fuller,B。(2011)。 使用无冷冻剂冷却器对包裹的肝球体进行低温保存:使用多步骤冷却进行高功能恢复个人资料。 Cryo Letters 32(2):158-165。
  8. Murua,A.,Orive,G.,Hernandez,R。M.和Pedraz,J.L。(2009)。 基于冷冻方案的冷冻保存,用于长期储存微囊化成肌细胞。 生物材料 30(20):3495-3501。
  9. Orive,G.,Santos,E.,Pedraz,J。L.和Hernandez,R。M.(2014)。 应用细胞包封控制生物治疗药物的递送。 Adv Drug Deliv Rev 67-68:3-14。
  10. Ruiz-Delgado GJ,Mancías-Guerra C,Tamez-GómezEL,Rodríguez-Romo LN,López-Otero A,Hernández-Arizpe A,Gómez-Almaguer D,Ruiz-ArgüellesGJ。 (2009年)。 脐带血干细胞移植中二甲基亚砜诱导的毒性:三例报告及文献复习。 Acta Haematol 122(1):1-5。
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引用:Gurruchaga, H., Saenz del Burgo, L., Orive, G., Hernandez, R. M., Ciriza, J. and Pedraz, J. L. (2019). Cell Microencapsulation and Cryopreservation with Low Molecular Weight Hyaluronan and Dimethyl Sulfoxide. Bio-protocol 9(4): e3164. DOI: 10.21769/BioProtoc.3164.
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