A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue
从软组织中制备富含生长因子的温敏性水凝胶方法   

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Acta Biomaterialia
Mar 2013

 

Abstract

Hydrogels are an ideal medium for the expansion of cells in three dimensions. The ability to induce cell expansion and differentiation in a controlled manner is a key goal in tissue engineering. Here we describe a detailed method for producing hydrogels from soft tissues with an emphasis on adipose tissue. In this method, soluble, extractable proteins are recovered from the tissue and stored while the remaining insoluble tissue is processed and solubilised. Once the tissue has been sufficiently solubilised, the extracted proteins are added. The resulting product is a thermosensitive hydrogel with proteins representative of the native tissue. This method addresses common issues encountered when working with some biomaterials, such as high lipid content, DNA contamination, and finding an appropriate sterilisation method. Although the focus of this article is on adipose tissue, using this method we have produced hydrogels from other soft tissues including muscle, liver, and cardiac tissue.

Keywords: Hydrogel (水凝胶), Adipose (脂肪), Soft tissue (软组织), Extracellular matrix (细胞外基质), Tissue engineering (组织工程), Biomaterial (生物材料), Method (方法), Protocol (方案)

Background

The main goal of tissue engineering is to generate new tissue by providing the body with a scaffold possessing similar properties to those of the target site. This allows for optimal remodeling and enables formation of de novo endogenous tissue. In the field of adipose tissue engineering, biomaterials derived from adipose tissue are of particular interest because adipose tissue is widely available and in theory provides the best possible environment for induction of adipogenesis (Flynn et al., 2007; Flynn, 2010; Uriel et al., 2008; Choi et al., 2009; Young et al., 2011). It has been established that adipocytes secrete adipogenic factors (Li et al., 1998; Shillabeer et al., 1989; Shillabeer et al., 1990) and that conditioned medium produced from either adipocytes or excised adipose tissue is able to induce adipogenesis in vitro (Sarkanen et al., 2012).

The development of an injectable hydrogel with properties closely matching those of healthy adipose tissue would potentially be of great use in regenerative medicine. The ideal hydrogel would be acellular, contain proteins that are representative of those found in natural adipose tissue, be structurally capable of maintaining a space after implantation, and be capable of inducing adipose tissue growth (Cheung et al., 2014; Drury and Mooney, 2003). We have previously reported on the production of a thermoresponsive hydrogel from excised adipose tissue which was shown to be adipogenic both in vitro and in vivo (Poon et al., 2013). The gel induced adipogenic differentiation of adipose-derived stem cells in vitro and was capable of producing adipose tissue from 8 weeks post-implantation in the subcutaneous layer of the rat back (Debels et al., 2015).

As originally reported, our adipose-derived hydrogel used dispase to decellularise the tissue prior to extraction. Although dispase is capable of efficiently decellularising tissue (Uriel et al., 2008; Prasertsung et al., 2008), we have since observed the degree of digestion varies greatly from batch to batch due to differences in tissue surface area. Additionally, the slight differences in decellularisation led to variations in lipid content between batches which altered protein extraction efficiency and clarity of the final product. The washing and delipidation steps were also of concern as they increased variation of the gel’s final physical properties. Since our original publication, we have developed a practical and efficient method to replace these early processes.

Here we provide a detailed protocol for producing a soft tissue-derived hydrogel which addresses many of these concerns and reduces batch-to-batch variability. In our new method, proteins are extracted from the tissue first in order to retain as much soluble protein as possible for subsequent re-addition. Decellularisation by dispase digestion has been replaced with cold homogenisation and nuclease treatment. Dispase is capable of efficiently decellularising tissue; it works by cleaving fibronectin and collagen IV, but there is a problem, these proteins may provide important functional groups which would otherwise be lost after dispase digestion (Gregoire et al., 1998; Khoshnoodi et al., 2008). Delipidation is no longer performed over the course of multiple salt washes and centrifugation steps, now it is done as part of the homogenisation and solubilisation steps. This method has been used to successfully produce thermoresponsive hydrogels from multiple tissues including skeletal muscle and organs such as the liver and heart. These hydrogels containing a collection of soluble proteins present in the native tissues may provide others in the field the basis for further developments in biomaterials research.

Materials and Reagents

  1. Whatman glass fibre filter paper (Sigma-Aldrich, catalog number: WHA1820021 )
  2. Spectrum Laboratories 12-14 kDa dialysis membrane (Pacific Laboratory Products, catalog number: 132706 )
  3. Freshly harvested subcutaneous porcine adipose tissue, liver, skin, cardiac tissue, and visceral fat (Donated by Diamond Valley Pork [Laverton North, VIC, Australia])
    Note: All tissue not used immediately was stored at -20 °C.
  4. Human tissues (Collected from fully consented patients with ethics approval from the St Vincent’s Hospital Melbourne Human Research Ethics Committee [Protocol 52/03])
    Note: All tissue not used immediately was stored at -20 °C.
  5. Protease inhibitors (Sigma-Aldrich)
  6. Ammonium sulphate (Sigma-Aldrich, catalog number: 31119 )
  7. 70% ethanol (Sigma-Aldrich)
  8. Spectrum Laboratories Spectra/Gel Absorbent (Pacific Laboratory Products, catalog number: 292600 )
  9. PBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
  10. DNeasy Blood & Tissue Kit (QIAGEN, catalog number: 69504 )
  11. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  12. Dulbecco’s modified Eagle medium (Sigma-Aldrich, catalog number: D5796 )
  13. Fetal calf serum (CSL, Australia)
  14. Antibiotics
  15. Oil Red O (Sigma-Aldrich, catalog number: O0625 )
  16. Haematoxylin
  17. Collagenase I
  18. 4% paraformaldehyde or 10% neutral-buffered formalin
  19. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: 798681 )
  20. N-ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: E3876 )
  21. Benzamidine hydrochloride hydrate (Sigma-Aldrich, catalog number: B6506 )
  22. Urea (Sigma-Aldrich, catalog number: U1250 )
  23. Guanidine hydrochloride (GuHCl) (Sigma-Aldrich, catalog number: 50950 )
  24. Glacial acetic acid (Sigma-Aldrich)
  25. Tris base (Sigma-Aldrich, catalog number: RDD008 )
  26. NaCl
  27. DNase I (Roche Diagnostics, catalog number: 11284932001 )
  28. RNase A (Roche Diagnostics, RNASEA-RO )
  29. MgCl2
  30. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251 )
  31. Chloroform (Sigma-Aldrich)
  32. Methanol (Sigma-Aldrich)
  33. Pepsin (Worthington Biochemical, catalog number: LS003317 )
  34. Solutions (Prepared according to the directions outlined in Recipes)
    1. 0.5 M ethylenediaminetetraacetic acid (EDTA)
    2. 50 mg/ml N-ethylmaleimide (NEM)
    3. 50 mg/ml benzamidine hydrochloride hydrate
    4. 8 M urea
    5. 8 M guanidine hydrochloride (GuHCl)
    6. 0.5 N acetic acid
    7. 4 M GuHCl extraction buffer
    8. 10x Tris-buffered saline (TBS)
    9. Nuclease solution
    10. 1,000x haemoglobin precipitation solution
    11. Chloroform-methanol lipid extraction solution
    12. 10x protease inhibitors
    13. 0.75% pepsin

Equipment

  1. Knife or scalpel
  2. Balance
  3. 4 L beaker
  4. Rotary mixer
  5. Shaking incubator
  6. Centrifuge (capable of speeds of 15,000 x g)
  7. Food processor, immersion blender, or high capacity tissue homogeniser
  8. Cheesecloth or fine mesh gauze
  9. Humidified 37 °C incubator with 5% CO2 (for in vitro testing)
  10. Fume hood

Software

  1. ImageJ (https://imagej.nih.gov/ij/)
  2. GraphPad Prism 6 (GraphPad Software, Inc.)

Procedure

  1. Dissection
    Using a sharp knife or scalpel, shave frozen tissue into 2-3 mm thick pieces and weigh on a balance. Freezing the tissue serves two purposes - to lyse the cells and to make slicing easier. Freshly harvested tissue may also be used but care must be taken to keep the tissue cold. The final yield of hydrogel will be 1-3x the volume of the tissue you are processing.
    Notes:
    1. Freshly collected tissue, whether animal or human, may be processed immediately or stored at -20 °C to -80 °C for longer term storage prior to processing. Please note that tissue stored for extended periods (> 6 months) will result in hydrogels with reduced efficacy.
    2. For convenience, the weight of the tissue in the following steps is referred to as one (1x) volume.

  2. Extraction
    1. Transfer the sliced tissue to an airtight container and add a minimum of 2x volumes (based on weight of tissue) of 4 M guanidine hydrochloride (GuHCl) extraction buffer. Gently mix at 4 °C for 16-48 h to allow for complete extraction of the tissue.
      Note: The volume of GuHCl extraction buffer used depends on the tissue being processed; tissue with a high level of extractable protein will require larger volumes. Adjust the volume of GuHCl as required. In general, if the resulting protein extract is highly viscous, add an additional volume of extraction buffer and incubate for another 16 h for further extraction – tissue very high in collagen such as skin and tendon will have lower amounts of total extractable protein than other tissue such as muscle and soft organs like liver. Ideally, extraction steps should be done in an end-over-end manner with a rotary mixer to ensure the tissue is properly mixed during incubation. Alternative mixers may also be used provided they result in adequate movement of the tissue during extraction. The use of magnetic stirrers is not recommended.
    2. After at least 16 h, collect the 4 M GuHCl containing the soluble protein extracted from the tissue and filter through glass fibre filter paper to remove particulate material.
      Note: If the 4 M GuHCl protein extract does not easily pass through glass fibre filter paper, dilute it with 4 M GuHCl and try again.
    3. Dialyse the protein extract against a total of 100x volumes of water at 4 °C.
      a. Pour the 4 M GuHCl protein extract to a suitable length of dialysis tubing, being sure not to fill the tubing more than ~70% to allow for expansion during dialysis.
      b. Transfer the dialysis tubing containing the protein extract to a pre-chilled 4 L beaker of water and mix at 4 °C.
      c. Replace the water every 1-2 h until a total of ~100x volumes of water has been used for dialysis (the dialysis may be left overnight).
    4. Take the leftover extracted tissue and wash in water for 1 h by gently mixing at 4 °C (change the water every 10 min). Once washed, begin processing this tissue as directed in the next section - Tissue processing.
      Note: After completing steps B2-B4, you will have two samples - a liquid GuHCl fraction containing extracted proteins, and leftover pieces of extracted tissue. The liquid fraction is processed according to instructions outlined in steps B5-B8; the solid tissue is processed according to step C - Tissue Processing.
    5. After dialysis in water, measure the volume of the GuHCl extract. Add 1 µl of 0.5 M zinc sulphate (ZnSO4) (1,000x stock) per ml of GuHCl extract, to begin precipitating haemoglobin (Hover and Kulkarni, 2000). Briefly mix, then incubate the protein extract at 4 °C for 30 min before centrifuging at 15,000 x g for 15 min at 4 °C.
    6. Transfer the supernatant to a clean container and add protease inhibitors (1 mM each of N-ethylmaleimide, benzamidine, and EDTA). Transfer the extract to dialysis membrane and dialyse overnight in water at 4 °C to remove excess ZnSO4
      1. Pour the extract to a suitable length of dialysis tubing, being sure not to fill the tubing more than ~70% to allow for expansion during dialysis.
      2. Transfer the dialysis tubing containing the protein extract to a pre-chilled 4 L beaker of water and mix at 4 °C.
      3. Replace the water every 1-2 h until a total of ~100x volumes of water has been used for dialysis (the dialysis may be left overnight).
      Note: The final working concentration of ZnSO4 is 0.5 mM. Steps B5-B6 may be skipped if there is no obvious haemoglobin contamination i.e., the protein extract has very little colour.
    7. After dialysis, measure the volume of the protein extract and transfer to a beaker with a capacity at least double that of the extract volume. While stirring the extract on ice, slowly dissolve 0.5 g of ammonium sulphate per ml of tissue extract (Doonan, 1996). Once the ammonium sulphate has completely dissolved, allow the protein to precipitate at 4 °C for at least 1 h or preferably overnight.
    8. Once precipitated, collect the protein by centrifugation at 15,000 x g for 20 min at 4 °C. Discard the supernatant and dissolve the protein pellet in water. Use as little water as possible to completely dissolve the protein. Store at -20 °C until step D (final processing).
      Note: The residual ammonium sulphate present in the pellet will help solubilise the protein. Although extracts stored for up to 12 months have been shown to retain much of their efficacy, it is preferable to limit the storage time prior to use. 

  3. Tissue processing
    1. After washing the pieces of GuHCl-extracted tissue in water, re-weigh the tissue, then transfer to a large container and homogenise in an excess volume of chilled water (> 20x volumes).
      Note: High capacity tissue homogenisers are difficult to find; commercially available food processors or immersion blenders may be used instead. If adipose tissue is being processed, it is important to thoroughly homogenise the tissue to release the lipid from the tissue. It is important that the water used in this step is chilled so that any lipid present in the tissue becomes solid.
    2. Using forceps, remove a small amount of tissue, rinse thoroughly in a separate beaker of water and transfer to a clean container on ice (Figure 1). Repeat this process until all of the tissue has been collected. At this stage, it may be necessary to repeat this procedure if you are working with adipose tissue - homogenise in chilled water and repeat as necessary until all visible lipid is removed.


      Figure 1. Procedure for cleaning homogenised tissue

    3. Once washed, blot the tissue dry against absorbent paper and measure the weight - this is 1x volume.
    4. Prepare 1.5x volumes of nuclease buffer. Transfer the tissue and nuclease buffer to an airtight container and gently mix at 37 °C for at least 1 h.
    5. After digestion, discard the nuclease buffer and wash the tissue three times in water at room temperature.
    6. Discard the water and add at least 10x volumes of 70% ethanol. Mix at 37 °C for at least 15 min. Discard the ethanol, then wash the tissue three times with water at room temperature.
    7. For adipose or similarly fatty tissues, the Folch method of lipid extraction may be used at this stage to further remove any remaining lipid (Folch et al., 1957). Prepare 10-20x volumes of 2:1 (chloroform:methanol) and mix with the tissue for 20 min in a fume hood at room temperature. Discard the liquid waste appropriately, then wash the tissue thoroughly three or more times with an excess volume of water. Blot the tissue dry against absorbent paper and measure the weight - this is 1x volume.
    8. Prepare 2x volumes of 0.75% pepsin in 0.5 M acetic acid. Mix at room temperature until completely dissolved. Rinse the tissue briefly (30 sec) with an excess volume of 0.5 M acetic acid to pre-swell the tissue. Discard the acetic acid, transfer the tissue to an air-tight container, and add the pepsin. Mix vigorously at room temperature until the tissue has been digested (≥ 2 days - check the digest regularly). Be sure to incubate the tissue at room temperature; not 37 °C, and do not digest the tissue longer than is necessary. The digested tissue should be relatively clear and quite viscous.
      Note: Alternatively, the tissue may be homogenised with an equal volume of 0.5 M acetic acid prior to adding the solubilised pepsin. This will greatly shorten the required digestion time, however the end product may require concentration before use. Depending on the tissue being digested, it may be necessary to add additional pepsin. Tissue very high in collagen such as skin and tendon may require the addition of more pepsin for complete digestion (Neuman et al., 1950). After 24 h, if the digest is especially viscous, additional 0.75% pepsin may be added until the tissue is able to be easily mixed. Care must be taken to only add the minimum amount of pepsin as necessary in order to reduce the potential for immunogenic reactions (Northrop, 1930; Samuels et al., 2009; Seastone et al., 1937). Mix until digested.
    9. Once digested, remove any particulate matter by squeezing through cheesecloth, fine mesh gauze or the equivalent. Follow this with centrifugation at 15,000 x g, 4 °C, 20 min. Carefully remove any visible lipid at the surface and transfer the digested protein to dialysis membrane. Dialyse against 100x volumes of chilled TBS at 4 °C for 16-48 h to neutralise the pepsin.
      Note: Use a spatula to collect all of the digested protein from the cheesecloth. A lot of protein can be lost at this stage if you are not careful.

  4. Final processing
    1. Remove the liquid protein extract from -20 °C storage and thaw. Add all of the liquid extract to the neutralised pepsin digest and mix well. Transfer the mixture to dialysis membrane and begin sterilisation. At this stage, Spectra/Gel Absorbent may be used at 4 °C to increase the concentration of the gel prior to sterilisation (following the manufacturer’s instructions).
    2. Sterilise the gel by dialysis in chilled 8 M urea (Roberts and Lloyd, 2007) overnight at 4 °C, followed by dialysis overnight in chilled 0.5% chloroform/TBS at 4 °C.
      Note: After dialysis in 8 M urea, all of the protein should be thoroughly solubilised and more concentrated.
    3. Finally, dialyse against 100x volumes of chilled TBS for 16 h and finish with dialysis against 100x volumes of chilled PBS for 16 h. Store aliquots of gel at -20 °C to -80 °C. Working aliquots of hydrogel should be thawed slowly at 4 °C. Thawed hydrogels may be stored at 4 °C for one month without any loss of efficacy. Depending on the desired concentration, the final yield should be between 1-3x the starting volume of tissue. A sample of the final product should be assayed for DNA (QIAGEN DNeasy Blood & Tissue Kit) and protein concentration (PierceTM BCA Protein Assay Kit) before undergoing in vitro testing. Gelation should occur within 10 min at 37 °C. Sterile PBS may be used to adjust the final protein concentration if desired.
      Note: Protein concentrations > 3 mg/ml are recommended to maintain integrity of the hydrogel after gelation, however gelation will occur at concentrations ~1 mg/ml.

Data analysis

  1. The manner in which the hydrogels are tested and analysed is entirely up to the discretion of the researcher, however the type of cells used for testing, the number of replicates, and the total number of cells used should be sufficient to give meaningful, consistent results. The general procedure described below for adipose-derived hydrogel testing may be adapted for the testing of other types of tissue-derived hydrogels.
  2. The following section expands on, and supplements, the analysis originally described in our previous article for the analysis of the adipogenic activity of adipose-derived hydrogels (Poon et al., 2013); the reader is advised to refer to that publication for experimental methodology and further details.
  3. In vitro-testing of adipogenic hydrogel is typically carried out using adipose-derived stem cells (ADSC) cultured in Dulbecco’s modified Eagle medium supplemented with 10% foetal calf serum and antibiotics. Cells are then either supplemented with a cocktail of adipogenic components (Zuk et al., 2001) or with the hydrogel test material. After a period of approximately two weeks, the hydrogel is removed from the culture dishes prior to fixation and staining with Oil Red O (90 min) and haematoxylin (3 min). In the cases where hydrogels are difficult to remove, a short digestion with collagenase I (2 mg/ml) will help to detach the gels. After thoroughly removing all traces of hydrogel from the culture dishes, the cells are fixed in formaldehyde (4% paraformaldehyde or 10% neutral-buffered formalin), stained with Oil Red O/haematoxylin, and then quantitated.
    Note: Hydrogels can be pre-set on one edge of the tissue culture dish prior to cell seeding – pipette a volume of hydrogel in the dish and incubate at 37 °C at a 45° angle until the gel has fully set (~10 min).
  4. Quantitation of the differentiated cells in our original publication was performed using the freely available software, ImageJ (https://imagej.nih.gov/ij/) (Rasband, 1997-2016; Abramoff et al., 2004).
  5. If the tissue culture wells are sufficiently small, or your photographic equipment is capable, it is advised to take a single, high resolution image of the entire culture surface; otherwise multiple randomly-selected fields should be photographed for each tissue culture well.
  6. Each of the images is opened in ImageJ and analysed as follows:
    1. Open image in ImageJ.
    2. Select Plugins→Analyze→Grid. Choose an appropriate colour for the grid lines and select OK (Figure 2).


      Figure 2. Turning on Grid lines

    3. Select Plugins→Analyze→Cell Counter (Figure 3).


      Figure 3. Selecting the Cell Counter

    4. In the Cell Counter window that appears, select ‘Initialize’, then select one of the 8 counters. Each counter has a different colour (Figure 4).


      Figure 4. The Cell Counter window

    5. Start by clicking on all of the haematoxylin-stained cells to get a total cell count, a running tally will appear in the window next to the counter number. Now choose another counter and click on all of the Oil Red O-stained cells.
    6. Once you have finished your counting, select Save Markers from the window to save your results.
    7. The proportion of Oil Red O-stained cells can be determined from these two numbers.
  7. After quantitating all of the images taken from the in vitro analysis, a table of figures can be produced for analysis. The numbers shown in Table 1 were the result of quantitation done for our previous publication (Poon et al., 2013). Statistical analysis by one way analysis of variance with post-hoc Tukey’s multiple comparisons test was performed using GraphPad Prism 6 (GraphPad Software, Inc.) to compare the adipogenic activity of each test condition.

    Table 1. Quantitation of adipocytes. Human ADSCs were supplemented with adipogenic medium (Zuk), adipose-derived hydrogel (Gel), or adipogenic medium and adipose-derived hydrogel (Gel + Zuk), then stained with Oil Red O and haematoxylin, and quantitated.


    The final result of the in vitro analysis is shown in Table 2 and Figure 5 below; a version of this figure was presented previously (Poon et al., 2013).

    Table 2. Summary of statistical analysis by one way ANOVA with post-hoc Tukey’s multiple comparisons test



    Figure 5. The adipogenic activity of adipose-derived hydrogel versus Zuk’s adipogenic medium on human adipose-derived stem cells. The data shows the mean with SD. Statistical analysis by one way ANOVA shows a statistically significant increase in adipogenic activity when Zuk’s adipogenic medium is supplemented with adipose-derived hydrogel (*). This analysis was described previously (Poon et al., 2013).

    With adipose-derived hydrogels, we have observed some batch-to-batch variation in adipogenic activity. The variables responsible for this are unknown, but age appears to be an important factor. If possible, it is advisable to screen fat samples from multiple tissue sources for adipogenic activity prior to large scale production. Conditioned medium can be produced from tissue samples by incubating the fat in ten volumes of complete medium under standard tissue culture conditions for 24 h. Once filter-sterilised, the conditioned medium can be tested for adipogenic activity on ADSCs. The most adipogenic tissue as determined by this screening process can be used for hydrogel production.

Recipes

  1. 0.5 M ethylenediaminetetraacetic acid (EDTA) (250 ml)
    36.53 g of EDTA
    Adjust pH to 8
    Add double distilled water to 250 ml
  2. 50 mg/ml N-ethylmaleimide (NEM) (2.5 M; 10 ml)
    0.5 g of NEM
    Ethanol to 10 ml
  3. 50 mg/ml benzamidine hydrochloride hydrate (3.13 M; 1 ml)
    50 mg of benzamidine HCl
    Add double distilled water to 1 ml
  4. 8 M urea (1 L)
    480.48 g of urea
    Add double distilled water to 1 L
    Filter through glass fibre filter paper
  5. 8 M guanidine hydrochloride (GuHCl) (1 L)
    764.32 g of GuHCl
    Add double distilled water to 1 L
    Filter through glass fibre filter paper
  6. 0.5 N acetic acid (1 L)
    971.2 ml of double distilled water
    28.8 ml of glacial acetic acid
  7. 4 M GuHCl extraction buffer (50 mM Tris-HCl pH 7.4; 4 M GuHCl; 100 ml)
    5 ml of 1 M Tris-HCl pH 7.4
    50 ml of 8 M GuHCl
    Add double distilled water to 100 ml
  8. 10x Tris-buffered saline (TBS) (0.5 M Tris-HCl; 1.5 M NaCl; 1 L)
    60.57 g of Tris
    87.66 g of NaCl
    Adjust pH to 7.4
    Add double distilled water to 1 L
    Filter through glass fibre filter paper
  9. Nuclease solution (50 µg/ml DNase I; 10 µg/ml RNase A; 10 mM MgCl2; 10 ml)
    50 µl of 10 mg/ml DNase I
    10 µl of 10 mg/ml RNase A
    100 µl of 1 M MgCl2
    PBS to 10 ml
  10. 1,000x haemoglobin precipitation solution (0.5 M ZnSO4; 50 ml)
    7.2 g of ZnSO4·7H2O
    Add double distilled water to 50 ml
  11. Chloroform-methanol lipid extraction solution ([2:1] chloroform:methanol; 150 ml)
    100 ml of chloroform
    50 ml of methanol
  12. 10x protease inhibitors (10 mM EDTA pH 8; 10 mM N-ethylmaleimide; 10 mM benzamidine; 100 ml)
    2 ml of 0.5 M EDTA pH 8
    400 µl of 50 mg/ml NEM
    319 µl of 50 mg/ml benzamidine HCl
    Add double distilled water to 100 ml
  13. 0.75% pepsin (7.5 mg/ml pepsin; 50 ml)
    375 mg of pepsin
    0.5 M acetic acid to 50 ml

Acknowledgments

We thank Dr. Kiryu K. Yap (St Vincent’s Institute and University of Melbourne, Department of Surgery) for editorial assistance with the manuscript. This work was supported by the Victorian State Government OIS Program and National Health and Medical Research Council Project Grant 1064786.

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  24. Uriel, S., Huang, J. J., Moya, M. L., Francis, M. E., Wang, R., Chang, S. Y., Cheng, M. H. and Brey, E. M. (2008). The role of adipose protein derived hydrogels in adipogenesis. Biomaterials 29(27): 3712-3719.
  25. Young, D. A., Ibrahim, D. O., Hu, D. and Christman, K. L. (2011). Injectable hydrogel scaffold from decellularized human lipoaspirate. Acta Biomater 7(3): 1040-1049.
  26. Zuk, P. A., Zhu, M., Mizuno, H. Huang, J., Futrell, J. W., Katz, A. J., Benhaim, P., Lorenz, H. P., Hedrick, M. H. (2001). Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2): 211-228.

简介

水凝胶是三维细胞扩增的理想媒介。以受控方式诱导细胞扩增和分化的能力是组织工程中的关键目标。在这里,我们描述了从软组织生产水凝胶的详细方法,重点是脂肪组织。在该方法中,可溶性可提取蛋白从组织中回收并储存,而剩余的不溶组织被加工和溶解。一旦组织被充分溶解,就加入提取的蛋白质。所得产物是具有代表天然组织的蛋白质的热敏水凝胶。这种方法解决了在使用某些生物材料时遇到的常见问题,例如高脂质含量,DNA污染和找到适当的灭菌方法。尽管本文的重点是脂肪组织,但使用这种方法,我们已经从其他软组织(包括肌肉,肝脏和心脏组织)中产生了水凝胶。

背景 组织工程的主要目标是通过向身体提供具有与目标部位相似性质的支架来产生新的组织。这允许最佳的重塑并且使得能够形成新生内源性组织。在脂肪组织工程领域,来自脂肪组织的生物材料是特别有意义的,因为脂肪组织广泛可用,并且在理论上为脂肪形成诱导提供了最佳可能的环境(Flynn等人,2007; Flynn,2010; Uriel等人,2008; Choi等人,2009; Young等人,2011)。已经确定脂肪细胞分泌脂肪形成因子(Li et al。,1998; Shillabeer等人,1989; Shillabeer等人, 1990),并且由脂肪细胞或切除的脂肪组织产生的条件培养基能够在体外诱导脂肪形成(Sarkanen等人,2012)。
 开发具有与健康脂肪组织密切匹配的特征的注射用水凝胶可能在再生医学中有很大的用途。理想的水凝胶将是无细胞的,含有代表在天然脂肪组织中发现的蛋白质,在结构上能够在植入后保持空间,并且能够诱导脂肪组织生长(Cheung等人)。 ,2014; Drury和Mooney,2003)。我们之前已经报道了从切除的脂肪组织产生的热反应性水凝胶,其被体外表现为在体内成脂肪(< em>>(Poon等人,2013)。凝胶在体外诱导脂肪来源的干细胞的脂肪形成分化,并且能够在植入后8周在大鼠背部的皮下层产生脂肪组织(Debels等人,2015)。
 据最初报道,我们的脂肪来源的水凝胶在提取前使用分散酶去细胞组织。虽然分散能够有效地去除组织细胞(Uriel等人,2008; Prasertsung等人,2008),我们已经观察到消化程度从批次变化很大由于组织表面积的差异而批量化。此外,脱细胞化的轻微差异导致批次之间的脂质含量的变化,这改变了蛋白质提取效率和最终产品的清晰度。洗涤和脱脂步骤也受到关注,因为它们增加了凝胶的最终物理性能的变化。自从我们的原始出版物以来,我们开发了一种实用而有效的方法来取代这些早期过程。
 这里我们提供了一个生产软组织衍生水凝胶的详细方案,解决了许多这些问题,并减少了批次间的变化。在我们的新方法中,首先从组织中提取蛋白质,以保留尽可能多的可溶性蛋白质用于随后的再添加。通过分解酶消化的脱细胞化已被冷均质化和核酸酶处理所取代。分散酶能够有效地脱细胞组织;它通过切割纤维连接蛋白和胶原蛋白IV起作用,但是存在问题,这些蛋白质可能提供重要的官能团,否则在分解消化后将会丢失(Gregoire等人,1998; Khoshnoodi等人al。,2008)。在多次盐洗涤和离心步骤的过程中不再进行脱胶,现在作为均化和增溶步骤的一部分进行。该方法已被用于成功地从多种组织(包括骨骼肌和器官如肝脏和心脏)产生热反应性水凝胶。含有存在于天然组织中的可溶性蛋白质的这些水凝胶可以为该领域的其他人提供生物材料研究的进一步发展的基础。

关键字:水凝胶, 脂肪, 软组织, 细胞外基质, 组织工程, 生物材料, 方法, 方案

材料和试剂

  1. Whatman玻璃纤维滤纸(Sigma-Aldrich,目录号:WHA1820021)
  2. Spectrum Laboratories 12-14 kDa透析膜(Pacific Laboratory Products,目录号:132706)
  3. 新鲜收获的皮下猪脂肪组织,肝脏,皮肤,心脏组织和内脏脂肪(由Diamond Valley Pork捐赠[Laverton North,VIC,Australia])
    注意:立即使用的所有组织都保存在-20°C。
  4. 人体组织(来自圣文森特医院墨尔本人类研究伦理委员会[议定书52/03])的完全同意的患者获得道德准则的患者
    注意:立即使用的所有组织都保存在-20°C。
  5. 蛋白酶抑制剂(Sigma-Aldrich)
  6. 硫酸铵(Sigma-Aldrich,目录号:31119)
  7. 70%乙醇(Sigma-Aldrich)
  8. Spectrum Laboratories Spectra/Gel Absorbent(Pacific Laboratory Products,目录号:292600)
  9. PBS(Thermo Fisher Scientific,Gibco TM ,目录号:10010023)
  10. DNeasy血&组织试剂盒(QIAGEN,目录号:69504)
  11. Pierce TM BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23225)
  12. Dulbecco改良的Eagle培养基(Sigma-Aldrich,目录号:D5796)
  13. 胎牛血清(CSL,澳大利亚)
  14. 抗生素
  15. 油红O(Sigma-Aldrich,目录号:O0625)
  16. 苏木精
  17. 胶原酶I
  18. 4%多聚甲醛或10%中性缓冲福尔马林
  19. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:798681)
  20. N-马来酰亚胺(NEM)(Sigma-Aldrich,目录号:E3876)
  21. 盐酸苄脒盐酸盐(Sigma-Aldrich,目录号:B6506)
  22. 尿素(Sigma-Aldrich,目录号:U1250)
  23. 胍盐酸盐(GuHCl)(Sigma-Aldrich,目录号:50950)
  24. 冰醋酸(Sigma-Aldrich)
  25. Tris碱(Sigma-Aldrich,目录号:RDD008)
  26. NaCl
  27. DNase I(Roche Diagnostics,目录号:11284932001)
  28. RNase A(Roche Diagnostics,RNASEA-RO)
  29. MgCl 2
  30. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Sigma-Aldrich,目录号:Z0251)
  31. 氯仿(Sigma-Aldrich)
  32. 甲醇(Sigma-Aldrich)
  33. Pepsin(Worthington Biochemical,目录号:LS003317)
  34. 解决方案(根据食谱概述的方向准备)
    1. 0.5M乙二胺四乙酸(EDTA)
    2. 50mg/ml N,N-乙基马来酰亚胺(NEM)
    3. 50毫克/毫升盐酸哒嗪盐酸盐
    4. 8 M尿素
    5. 8 M盐酸胍(GuHCl)
    6. 0.5 N乙酸
    7. 4 M GuHCl提取缓冲液
    8. 10倍Tris缓冲盐水(TBS)
    9. 核酸酶溶液
    10. 1,000x血红蛋白沉淀溶液
    11. 氯仿 - 甲醇脂质提取液
    12. 10x蛋白酶抑制剂
    13. 0.75%胃蛋白酶

设备

  1. 刀或手术刀
  2. 平衡
  3. 4升烧杯
  4. 旋转搅拌机
  5. 震动孵化器
  6. 离心机(能够达到15,000 x g 的速度)
  7. 食品加工机,浸入式搅拌机或高容量组织均质机
  8. 粗棉布或细网纱布
  9. 5%CO 2加湿的37℃培养箱(用于体外试验)
  10. 通风柜

软件

  1. ImageJ( https://imagej.nih.gov/ij/
  2. GraphPad Prism 6(GraphPad Software,Inc.)

程序

  1. 解剖
    使用锋利的刀或手术刀,将冷冻的组织刮成2-3毫米厚的块,称重。冷冻组织有两个目的 - 裂解细胞,使切片更容易。也可以使用新鲜收获的组织,但必须注意保持组织冷。水凝胶的最终产量将是正在加工的组织体积的1-3倍。
    注意:
    1. 新鲜收集的组织,无论是动物还是人类,可以立即加工或储存在-20°C至-80°C,以便在加工前长时间储存。请注意,长时间储存(> 6个月)的组织会导致效果降低的水凝胶。
    2. 为了方便起见,以下步骤中的组织的重量被称为一(1x)体积。

  2. 萃取
    1. 将切片的组织转移到密封的容器中,并加入4M盐酸胍(GuHCl)提取缓冲液的至少2x体积(基于组织的重量)。在4℃下轻轻混合16-48小时以允许完全提取组织。
      注意:使用的GuHCl提取缓冲液的体积取决于正在处理的组织;具有高水平可提取蛋白质的组织将需要更大的体积。根据需要调整GuHCl的体积。通常,如果得到的蛋白质提取物是高度粘稠的,加入额外体积的提取缓冲液并孵育另外16小时用于进一步提取 - 非常高的胶原蛋白如皮肤和腱组织将具有比其他组织更少量的可提取蛋白质如肌肉和软器官如肝脏。理想情况下,采用旋转混合器,以端到端的方式进行提取步骤,以确保组织在孵育期间适当混合。也可以使用替代的混合器,只要它们在提取期间导致组织的充分运动。不推荐使用磁力搅拌器。
    2. 至少16小时后,收集含有从组织提取的可溶性蛋白质的4M GuHCl,并通过玻璃纤维滤纸过滤以除去颗粒物质。
      注意:如果4 M GuHCl蛋白质提取物不容易通过玻璃纤维滤纸,用4 M GuHCl稀释,然后重试。
    3. 在4℃下将蛋白质提取物与总共100倍体积的水进行透析。
      a。将4M GuHCl蛋白质提取物倒入合适长度的透析管中,确保不要将管道填充超过70%以允许在透析期间膨胀。
      b。将含有蛋白质提取物的透析管转移到预冷却的4L水中,并在4℃下混合。
      c。每1-2小时更换一次水,直到总共使用约100倍体积的水进行透析(透析可能会留置过夜)。
    4. 取剩余的提取物,在4℃下轻轻混合(每10分钟更换一次),在水中洗涤1小时。一旦清洗,开始处理这个组织,如下一节所述 - 组织处理。
      注意:完成步骤B2-B4后,您将有两个样品 - 含有提取蛋白质的液体GuHCl部分和提取的组织的剩余部分。根据步骤B5-B8中概述的说明书处理液体馏分;根据步骤C - 组织处理处理固体组织。
    5. 在水中透析后,测量GuHCl提取物的体积。加入1μl0.5M硫酸锌(ZnSO 4)(1,000x储备)/ml GuHCl提取物,开始沉淀血红蛋白(Hover和Kulkarni,2000)。简单混合,然后在4℃温育蛋白质提取物30分钟,然后在4℃下以15,000xg离心15分钟。
    6. 将上清液转移到干净的容器中并加入蛋白酶抑制剂(1mM的N-乙基马来酰亚胺,苯甲脒和EDTA)。将提取物转移到透析膜上,并在4℃的水中透析过夜以除去过量的ZnSO 4。 
      1. 将提取物倒入适当长度的透析管,确保不要将管道填充超过70%以允许在透析期间膨胀。
      2. 将含有蛋白质提取物的透析管转移到预冷却的4L烧杯中,并在4℃下混合。
      3. 每1-2小时更换一次水,直到总共使用〜100倍体积的水进行透析(透析可能会留置过夜)。
      注意:ZnSO 4的最终工作浓度为0.5mM。如果没有明显的血红蛋白污染,即蛋白质提取物的颜色很少,可以跳过步骤B5-B6。
    7. 透析后,测量蛋白质提取物的体积,并转移到烧杯中,容量至少为提取物体积的两倍。在冰上搅拌提取物时,缓慢溶解0.5ml硫酸铵/ml组织提取物(Doonan,1996)。一旦硫酸铵完全溶解,使蛋白质在4℃下沉淀至少1小时,或优选过夜
    8. 一旦沉淀,通过在4℃以15,000×g离心20分钟收集蛋白质。弃去上清液并将蛋白质颗粒溶解在水中。使用尽可能少的水来完全溶解蛋白质。储存于-20°C直至步骤D(最终处理)。
      注意:沉淀中存在的残留硫酸铵将有助于溶解蛋白质。尽管存储的提取物长达12个月已被证明保留了很多功效,但最好在使用前限制储存时间。 

  3. 组织加工
    1. 在水中洗涤GuHCl提取的组织片后,重新称重组织,然后转移到大容器中,并在过量体积的冷冻水(> 20倍体积)中均质化。
      注意:高容量组织匀浆器很难找到;可以使用市售食品加工商或浸没式搅拌机。如果正在处理脂肪组织,重要的是彻底均匀组织以从组织释放脂质。重要的是,该步骤中使用的水被冷却,使得组织中存在的任何脂质变得固体。
    2. 使用镊子,取出少量的纸巾,在单独的烧杯中彻底冲洗,并转移到冰上的干净的容器(图1)。重复此过程,直到所有的组织都被收集。在这个阶段,如果您正在使用脂肪组织 - 在冷冻水中均质化并根据需要重复,直到所有可见的脂质被去除,可能需要重复此过程。


      图1.清洁均质组织的步骤

    3. 一旦洗涤,将纸巾干净吸干纸,并测量重量 - 这是1x体积。
    4. 准备1.5倍体积的核酸酶缓冲液。将组织和核酸酶缓冲液转移到密封容器中,并在37℃下轻轻混合至少1小时。
    5. 消化后,弃去核酸酶缓冲液,并在室温下将水洗三次。
    6. 弃去水并加入至少10倍体积的70%乙醇。在37℃下混合至少15分钟。丢弃乙醇,然后在室温下用水洗涤组织三次。
    7. 对于脂肪或类似的脂肪组织,可以在此阶段使用Folch脂质提取方法以进一步除去任何残留的脂质(Folch等人,1957)。准备10-20倍体积的2:1(氯仿:甲醇),并在室温下在通风橱中与组织混合20分钟。相应地丢弃液体废物,然后用过量的水彻底洗涤组织三次或更多次。将纸巾与吸收纸干燥并测量重量 - 这是1x体积。
    8. 在0.5M乙酸中制备2倍体积的0.75%胃蛋白酶。在室温下混合直到完全溶解。用过量体积的0.5M乙酸短暂洗涤组织(30秒)以预先膨胀组织。弃去乙酸,将组织转移到气密容器中,加入胃蛋白酶。在室温下剧烈混合,直到组织消化(≥2天 - 定期检查摘要)。确保在室温下孵育组织;不要37℃,不要长于需要消化组织。消化的组织应相对清澈,相当粘稠。
      注意:或者,可以在加入溶解的胃蛋白酶之前,用等体积的0.5M乙酸将组织匀浆。这将大大缩短所需的消化时间,但最终产品在使用前可能需要浓缩。根据正在消化的组织,可能需要加入额外的胃蛋白酶。胶原蛋白如皮肤和肌腱组织非常高,可能需要添加更多的胃蛋白酶才能完全消化(Neuman et al。,1950)。 24小时后,如果消化物特别粘稠,可以加入另外0.75%的胃蛋白酶,直到组织能够容易地混合。必须注意,只有添加最少量的胃蛋白酶才能降低免疫原性反应的可能性(Northrop,1930; Samuels et al。,2009; Seastone et al。,1937)。混合直至消化。
    9. 一旦消化,通过挤压通过粗棉布,细网纱或等效物去除任何颗粒物。按照15,000 x g,4℃,20分钟离心分离。小心地清除表面上的任何可见的脂质,并将消化的蛋白质转移到透析膜上。在4℃下对100x体积的冷冻TBS进行透析16-48小时以中和胃蛋白酶。
      注意:使用刮刀从干酪布中收集所有消化的蛋白质。如果不小心,很多蛋白质在这个阶段可能会丢失

  4. 最后处理
    1. 将液体蛋白质提取物从-20°C储存并解冻。将所有液体提取物加入到中和的胃蛋白酶消化物中并充分混合。将混合物转移到透析膜上并开始灭菌。在这个阶段,Spectra/Gel Absorbent可以在4℃下使用,以增加灭菌前的凝胶浓度(遵照制造商的说明)。
    2. 在冷冻的8M尿素(Roberts and Lloyd,2007)中透析在4℃下过夜灭菌凝胶,然后在4℃在冷却的0.5%氯仿/TBS中透析过夜。
      注意:在8M尿素透析后,所有蛋白质应彻底溶解并浓缩。
    3. 最后,对100x体积的冷冻TBS透析16小时,并完成对100倍体积的冷冻PBS透析16小时。在-20°C至-80°C下保存凝胶等分试样。水凝胶的工作等分试样应在4℃下缓慢解冻。解冻的水凝胶可以在4℃下储存1个月,不会有任何的功效丧失。取决于所需的浓度,最终产量应在组织起始体积的1-3倍之间。在进行体外测试之前,应测定最终产品的样品中的DNA(QIAGEN DNeasy Blood& Tissue Kit)和蛋白质浓度(Pierce TM BCA Protein Assay Kit) 。凝胶应在37℃下10分钟内进行。如果需要,可以使用无菌PBS调节最终的蛋白质浓度 注意:蛋白质浓度>建议使用3mg/ml以维持凝胶化后的水凝胶的完整性,然而凝胶化将在浓度为1mg/ml时发生。

数据分析

  1. 测试和分析水凝胶的方式完全由研究者决定,然而用于测试的细胞类型,重复次数和使用的细胞总数应足以产生有意义的一致结果。下面描述的脂肪来源的水凝胶测试的一般程序可以适用于其他类型的组织衍生的水凝胶的测试。
  2. 以下部分扩展和补充了我们以前的文章中描述的分析脂肪来源水凝胶的脂肪生成活性的分析(Poon等人,2013);建议读者参考该出版物的实验方法和进一步的细节。
  3. 脂肪形成水凝胶的体外测试通常使用在补充有10%胎牛血清和抗生素的Dulbecco改良的Eagle培养基中培养的脂肪来源的干细胞(ADSC)来进行。然后将细胞补充有脂肪形成组分的混合物(Zuk等人,2001)或与水凝胶测试材料。在约两周的时间之后,在固定之前将水凝胶从培养皿中取出,并用油红O(90分钟)和苏木精(3分钟)染色。在水凝胶难以除去的情况下,用胶原酶I(2mg/ml)进行短消化有助于分离凝胶。从培养皿中彻底清除所有痕量的水凝胶后,将细胞固定在甲醛(4%多聚甲醛或10%中性缓冲福尔马林)中,用油红O /苏木精染色,然后定量。 注意:在细胞接种之前,水凝胶可以预先设置在组织培养皿的一个边缘上 - 将一定体积的水凝胶移至培养皿中,并在37℃以45度角孵育直到凝胶完全凝固( 〜10分钟)。
  4. 使用免费提供的软件ImageJ()进行原始出版物中的分化细胞的定量https://imagej.nih.gov/ij/)(Rasband,1997-2016; Abramoff等人,2004)。
  5. 如果组织培养孔足够小,或者您的摄影设备有效,建议采用整个培养面的单一高分辨率图像;否则应为每个组织培养井拍摄多个随机选择的场。
  6. 每个图像在ImageJ中打开并分析如下:
    1. 在ImageJ中打开图像。
    2. 选择插件→分析→网格。为网格线选择合适的颜色,然后选择OK(图2)。


      图2.打开网格线

    3. 选择插件→分析→单元格计数器(图3)。


      图3.选择单元格计数器

    4. 在出现的单元格计数器窗口中,选择"初始化",然后选择8个计数器之一。每个计数器都有不同的颜色(图4)

      图4.单元格计数器窗口

    5. 首先点击所有苏木精染色的细胞以获得总细胞数,运行的计数将出现在计数器号旁边的窗口中。现在选择另一个计数器,然后点击所有的油红O染色细胞。
    6. 完成计数后,从窗口中选择保存标记以保存结果。
    7. 油红O染色细胞的比例可以从这两个数字确定。
  7. 在对从体外分析获得的所有图像进行定量后,可以生成一张图表以进行分析。表1中显示的数字是我们以前的出版物(Poon等人,2013年)的定量结果。使用GraphPad Prism 6(GraphPad Software,Inc。)进行单因素方差分析与事后Tukey多重比较检验的统计分析,以比较每个测试条件的成脂活性。

    表1.脂肪细胞定量人ADSCs补充有脂肪生成培养基(Zuk),脂肪来源的水凝胶(Gel)或脂肪生成培养基和脂肪来源的水凝胶(Gel + Zuk),然后用油红O和苏木精,定量。


    体外分析的最终结果示于下表2和图5中。这个数字的一个版本以前提出(Poon等人,2013)。

    表2.通过单因素统计分析的总结ANOVA与事后Tukey的多重比较测试



    图5.脂肪来源的水凝胶与Zuk的脂肪形成培养基对人脂肪干细胞的脂肪生成活性。数据显示SD的平均值。统计学分析方法ANOVA显示脂肪生成活性的统计学显着增加,当Zuk的脂肪生成培养基补充脂肪来源的水凝胶(*)。以前描述了这种分析(Poon等人,2013)。

    对于脂肪来源的水凝胶,我们观察到脂肪生成活性的一批间批次变化。负责这个的变量是未知的,但年龄似乎是一个重要因素。如果可能,建议在大规模生产之前筛选多个组织来源的脂肪样品用于脂肪生成活性。通过在标准组织培养条件下将十倍体积的完全培养基中的脂肪温育24小时,可以从组织样品产生条件培养基。一旦过滤灭菌,可以测试条件培养基对ADSC的脂肪生成活性。通过该筛选方法测定的最成脂组织可用于水凝胶生产。

食谱

  1. 0.5M乙二胺四乙酸(EDTA)(250ml)
    36.53克EDTA
    将pH调节至8
    加入双蒸水至250 ml
  2. 50mg/ml N,N-乙基马来酰亚胺(NEM)(2.5M; 10ml)
    0.5克NEM
    乙醇至10 ml
  3. 50mg/ml苄脒盐酸盐水合物(3.13M; 1ml)
    50毫克苯甲脒盐酸盐 加入双蒸水至1 ml
  4. 8 M尿素(1升)
    480.48克尿素
    加入双蒸水至1升 过滤玻璃纤维滤纸
  5. 8M盐酸胍(GuHCl)(1L)
    764.32 g的GuHCl
    加入双蒸水至1升 过滤玻璃纤维滤纸
  6. 0.5 N乙酸(1升)
    971.2ml双蒸水
    28.8ml冰醋酸
  7. 4M GuHCl提取缓冲液(50mM Tris-HCl pH7.4; 4M GuHCl; 100ml) 5毫升1M Tris-HCl pH7.4
    50ml 8M GuHCl
    加入双蒸水至100ml
  8. 10倍Tris缓冲盐水(TBS)(0.5M Tris-HCl; 1.5M NaCl; 1L) 60.57克Tris
    87.66克NaCl
    将pH调节至7.4
    加入双蒸水至1升 过滤玻璃纤维滤纸
  9. 核酸酶溶液(50μg/ml DNA酶I;10μg/ml核糖核酸酶A; 10mM MgCl 2; 10 ml)
    50μl10 mg/ml DNase I
    10μl10 mg/ml RNase A
    100μl1M MgCl 2
    PBS至10 ml
  10. 1,000x血红蛋白沉淀溶液(0.5M ZnSO 4·50ml)
    7.2g ZnSO 4·7H 2 O
    加入双蒸水至50 ml
  11. 氯仿 - 甲醇脂质提取溶液([2:1]氯仿:甲醇; 150ml) 100ml氯仿
    50ml甲醇
  12. 10倍蛋白酶抑制剂(10mM EDTA pH8; 10mM N,N-乙基马来酰亚胺; 10mM苄脒; 100毫升)
    2 ml 0.5 M EDTA pH 8
    400μl50 mg/ml NEM
    319μl50 mg/ml盐酸苯甲脒
    加入双蒸水至100ml
  13. 0.75%胃蛋白酶(7.5mg/ml胃蛋白酶; 50ml)
    375毫克胃蛋白酶
    0.5 M乙酸至50 ml

致谢

我们感谢Kiryu K. Yap博士(圣文森特研究所和墨尔本大学外科系)对稿件的编辑帮助。维多利亚州政府OIS计划和国家卫生和医学研究委员会项目拨款1064786支持这项工作。

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引用:Poon, C. J., Tan, S. S., Boodhun, S. W., Abberton, K. M. and Morrison, W. A. (2017). A Streamlined Method for the Preparation of Growth Factor-enriched Thermosensitive Hydrogels from Soft Tissue. Bio-protocol 7(3): e2128. DOI: 10.21769/BioProtoc.2128.
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