ひと腎臓血管周囲間質細胞の新規臨床等級分離法

The local medical ethical committee and ethical advisory board of the European consortium (STELLAR) approved the research and collection of human transplant grade kidneys discarded mainly for surgical reasons. Research consent was given for all kidneys. 1. Preparations for Cell Culture Prepare a pool of platelet lysates. Store the platelets that have expired for less than 2 d in -80 °C until use (for a maximum of 1 year). NOTE: The platelets were originally sourced from a commercial vendor. Hospital surplus material distributed by the local blood bank were obtained. Thaw the platelets of at least 5 donors (preferably 10) O/N at 4 °C. Pool the platelets in a large sterile bottle, transfer to conical centrifuge tubes and spin down for 10 min at 1,960 x g at 4 °C. NOTE: The volume of platelets depends on the number of donors and the amount of hospital surplus material per donor. Pipette the supernatant to blood bags (50 mL/bag) through the barrel of a 50 mL syringe. Store at -80 °C. Preparation of cell culture medium. Defrost one bag of platelet lysates in a water bath at 37 °C. Add 1 L (i.e. 2 bottles) of Minimum Essential Medium – alpha modification (alphaMEM), 5 mL 200 mM glutamine, and 20 mL of penicillin (5,000 U/mL)/streptomycin (5,000 µg/mL) (pen/strep) to the bag. Incubate for 3 h at 37 °C. Shake the bag for degelling by gently tapping on the bag when the bag is resting on a flat surface. Filter the 5% platelet lysates medium by pulling the lysates through the filter of the transfusion system with a sterile 50 mL syringe. Aliquot the medium in 50 mL tubes and store in -80 °C until use. NOTE: Every new batch of platelet lysates is tested in cell culture and compared to the previous batch by growing cells of interest (bmMSCs or kPSCs) to confluency in both batches. In cell numbers and viability, the kPSCs or bmMCs grown in the new batch should not differ more than 10%, and the marker expression of CD73, CD90, CD105 and CD31, CD34 and CD45 as analyzed by flow cytometry should not differ. The methods of cell culture are described in section 6 of the protocol (Culture of kPSC). 2. Preparations for Cell Harvest Preparation of solutions. Prepare the plain medium by adding 10 mL of pen/strep to 500 mL (1 bottle) of Dulbecco's Minimum Essential Medium (DMEM)-F12. Prepare the washing medium by adding 50 mL of Normal Human Serum (NHS) and 10 mL of pen/strep to 1 bottle of DMEM-F12. Prepare the enzyme-stock buffer by adding 2.9 mL 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.2 mL NaHCO3 2.5% [w/v] and 160 µL CaCl2 (1 M) to 160 mL University of Wisconsin solution. Prepare the collagenase solution by slowly adding 20 mL of enzyme stock buffer to the bottle of GMP-grade collagenase containing >2,000 U/bottle collagenase. Dissolve at 4 °C for approximately 40 min. Gently shake several times during the dissolving period. Prepare the freeze medium by adding 5 mL dimethyl sulfoxide (DMSO) to 45 mL NHS. Preparation of perfusion tray (Figure 1A). Clean the flow cabin and cover with surgical drapes. Heat a water bath to approximately 40 – 45 °C to heat the perfusion tray to 37 °C. Put on surgical gowns and surgical gloves. Connect a sterilized perfusion tray to the water bath to preheat the perfusion tray with warm water. Make sure that there is no air in the perfusion tray by starting with the perfusion tray in the vertical position. If necessary add more water to the water bath to prevent air infusion into the perfusion tray. Place the LS25 tube in the pump. Leave both sterile ends of the tube in the flow cabinet. Place a Kocher's forceps on one end of the tube and allow to rest on the bottom of the perfusion tray. Place a sterile gauze on top of the tube to prevent obstruction. Attach a Luer connector on the other tube end. Add approximately 100 mL of plain medium into the perfusion tray and start flushing the tube on a pump speed of 100 mL/min. 3. Kidney Cell Isolation Preparation of the kidney. Remove the kidney from the double sterile bags and place on the sterile perfusion tray (Figure 1B). Remove the perirenal adipose tissue and kidney capsule with scissors and gentle tearing (Figure 1C) and identify the renal artery on the aortic patch as shown in Figure 1D. Cannulate the renal artery with the accessory spike, fix with a sterilized tie-rip, and attach to the pump tube end with the Luer connector while the pump is on. If the tubes are not completely filled, first add more medium to the tray and flush tubes by starting the pump (Figure 1E – F). Flush the kidney with plain medium via the pump with a flow of 100 mL/min. Collagenase treatment and kidney cell isolation. Add collagenase (20 mL, >2,000 U) and DNase (2.5 mL, 1 mg/mL) to the perfusion tray (Figure 1G). Turn the kidney from time to time gently by hand. Wait until the kidney becomes soft and the perfusion liquid becomes less transparent (approximately 30 – 40 min). Gently massage the kidney (Figure 1H) until a cell suspension is obtained (Figure 1I). Remove non-digested material and the spike in the renal artery. Washing and collection of kidney cells. Add 50 mL of the NHS to the cell suspension. Drain the cell suspension from the perfusion tray by putting the pump at low flow and placing the tube first connected to the kidney above the 50 mL tubes (Figure 1J). Centrifuge at 300 x g for 7 min at 4 ˚C and remove the supernatant. Wash the pellets with washing medium containing 10% NHS and again centrifuge at 300 x g for 7 min. Repeat washing once more. Measure the volume of the cell pellets and add an equal volume of cell culture medium. Either cryopreserve the cells from here by adding freezing medium 1:1 to the cell suspension and store in cryogenic vials according to standard protocols, or continue to culture the cells (section 4). NOTE: The cell suspension contains cell clumps and requires extra steps to obtain single cells, which results in an increase in cell death. Therefore, the cells are kept in suspension and are not counted at this stage. 4. Cell Culture of Crude Kidney Cells Add approximately 1 mL of the cell suspension to 24 mL of alphaMEM 5% platelet lysates (cell culture medium) in a T175 cell culture flask (Figure 1K) and culture at 37 °C, 5% CO2. Remove the medium and cell debris after 2 d from the adherent cells and refresh the medium with 25 mL of cell culture medium. Refresh the culture medium twice a week by removing half of the medium (12.5 mL) and adding fresh medium (12.5 mL) using aseptic techniques. Culture until confluent (usually 5 – 7 d) (Figure 1L). Remove the medium, wash twice with PBS and trypsinize the cells by adding 5 mL trypsin to each T175 flask for 5 min at 37 °C. Wash in culture medium and centrifuge at 490 x g for 5 min and remove the supernatant. 5. Cell Enrichment for NG2 by Magnetic Cell Separation (Figure 1M) Prepare the cell separation buffer according to manufacturer's protocol. Resuspend the trypsinized kPSCs in 10 mL cell separation buffer. Centrifuge at 490 x g for 5 min, discard the supernatant and resuspend the cells in 300 µL cell separation buffer. Add 100 µL FcR blocking reagen/5 x 107 cells, add 100 µL anti-melanoma (NG2) beads per 5 x 107 cells, and incubate for 30 min at 4 °C. Separate the NG2-positive fraction according to manufacturer's protocol. Add 10 mL of medium and centrifuge cells for 5 min at 490 x g. Count the cells using a Bürker counting chamber according to manufacturer's protocol. Seed the cells in a culture flask (approximately 4 x 103 cells/cm2) and incubate. NOTE: Seed the cells in the following concentrations: <250,000 in a T25 flask, 250,000 – 500,000 in a T75 flask, and 700,000 – 800,000 in a T175 flask. After magnetic cell enrichment, a positive fraction of approximately 1 – 2% is isolated. However, as this step is cell enrichment and not cell sorting, other cell types will be present in the culture (Figure 1N). After several passages (usually around 4 – 5), only a homogenous kPSC-population is present (Figure 1P). 6. Culture of kPSCs Refresh the medium twice a week by removing half of the medium and replace it with freshly thawed culture medium (Figure 1N). NOTE: The kPSCs tend to be more viable and proliferative when only half of the medium is refreshed. Passaging of kPSCs. NOTE: Passage the kPSCs at either 90 – 100% confluency, or when 3D structures appear (see Figure 1O), or when there hasn't been cell growth for more than a week. The latter two particularly might occur at early passages after cell enrichment. In this case, after passaging, the cells will usually start to grow again in monolayer culture (Figure 1P). Remove the medium from the 90 – 100% confluent cells (keep the medium for later use). Wash the flask twice with PBS (1 mL in T25, 5 mL in T75, 10 mL in T175). Add trypsin (0.5 mL in T25, 2 mL in T75, 5 mL in T175) and incubate for 5 min at 37 °C. Add the old medium to the flask and resuspend gently. Transfer to a 15 or 50 mL tube (depending on the volume) and centrifuge at 490 x g for 5 min. Count the viable cells and plate <250,000 in a T25, 250,000 – 500,000 in a T75, or 700,000 – 800,000 in a T175 (approximately 4 x 103 cells/cm2). Test the kPSCs at passage 5 or 6 (e.g., scratch assay, section 7). NOTE: Characterize the propagated cells by flow cytometry using standard protocols. Marker expression of NG2, PDGFR-β, CD146, CD73, CD90, CD105, HLA-ABC should all be positive and CD31, CD34, CD45 and HLA-DR should be negative. Test for mycoplasma, bacteria and fungi in the culture medium according to standard protocols of the clinical microbiology lab. Test the kPSCs functionally in a kidney epithelial wound scratch assay. Experiments are usually performed with sterile, flow cytometry-confirmed homogeneous kPSC populations with wound healing capacity between passage 6-8. Cryopreserve the kPSCs by adding 0.5 mL cryopreservation medium to 0.5 mL cell suspension (2 million cells per mL of culture medium) using standard protocols. NOTE: After thawing, seed the kPSCs at a higher density (106 cells in a T175). 7. Functional Test of kPSCs: Kidney Epithelial Wound Scratch Assay Culture the kPSCs in a 6-well plate at a density of 200,000 cells/well in 2 mL culture medium. After 48 h of culture at 37 °C, 5% CO2, collect the supernatant. This is the conditioned medium. Seed the HK-2 (proximal tubular epithelial cells)17 in 2 mL of Proximal Tubular Epithelial Cell (PTEC) medium consisting of a 1:1 ratio of DMEM-F12 supplemented with insulin (5 mg/mL), transferrin (5 mg/mL), selenium (5 ng/mL), hydrocortisone (36 ng/mL), triiodothyrinine (40 pg/mL), epidermal growth factor (10 ng/mL) and pen/strep, in a density of 500,000 cells per well in a 6-well cell culture plate. Culture for 48 h at 37 °C, 5% CO2. Remove the cell culture medium of the HK-2s and create a scratch wound in the monolayer of HK-2s by making a scratch with the tip of a yellow pipette point from the top to the bottom of the well. Wash the HK-2s with PBS and add the conditioned medium of the kPSCs or control medium. Mark on the bottom of the plate the area to be imaged. NOTE: The HK-2s should be confluent. Image two areas per well. Image the scratch at 4, 8, 12 and 24 h at the same marked position with an inverted bright-field microscope. Measure the scratch area in Image J using the polygon selection tool to determine the percentage of closure.