人类肾脏血管周围间质细胞新型临床分级分离方法

Summary

在这里我们提出肾血管周围间质细胞 (kPSCs) 基于整个器官灌注与消化酶和 NG2 细胞丰富新颖的临床分级分离和培养方法。使用此方法，就可以获得足够的细胞数量，为细胞疗法。

Abstract

骨髓间质细胞 (MSCs) 是组织稳态和免疫调节细胞，有有利影响肾脏疾病和示移植。血管周围间质细胞 (Psc) 与骨髓间充质干细胞 （骨髓间充质干细胞） 共享特性。然而，他们也拥有，最有可能由于当地印迹、 组织特定的属性和地方组织的平衡中起着作用。这个组织特异性可能会导致组织具体的修复，还在人体肾脏内。我们先前表明人类肾脏物业服务公司 (kPSCs) 有增强肾上皮伤口愈合而骨髓间充质干细胞并没有这种潜力。此外，kPSCs 可以改善肾损伤的体内。因此，kPSCs 构成一个有趣的来源，细胞疗法，特别是对肾脏病和肾移植。在这里我们展示从移植级 kPSCs 详细的分离和培养方法基于整个器官灌注的消化酶通过肾动脉和血管周围标记 NG2 浓缩的人类肾脏。这种方式，适合细胞疗法可以获得大细胞数量。

Introduction

骨髓间质细胞 (MSCs) 是最初分离大鼠骨髓细胞的免疫调节细胞。他们的特点是其梭形形态，能够分化成脂肪、 骨和软骨及塑料粘附。骨髓间充质干细胞表达基质标记 CD73，CD90、 CD105 同时被标记 CD31 和 CD45^1，，23负。骨髓间充质干细胞是细胞疗法由于其组织稳态和免疫调节能力有前途的候选人。目前正在进行临床试验的几种疾病，包括肾脏病和肾移植在其他地方审查⁴研究骨髓间充质干细胞。

以前它是显示血管周细胞从几个不同的实体器官，包括脂肪、 胎盘和骨骼肌细胞与骨髓间充质干细胞⁹共享特性。但是，这些细胞也表现出组织特异性功能从而导致器官修复¹⁰。人类心肌血管周围的细胞，例如，刺激血管反应后缺氧和分化成心肌细胞，而从其他组织血管周细胞并没有表现出这些潜力¹¹。

血管周围间质细胞也可以隔绝鼠标^12，，1314和¹⁶人肾^15，。我们广泛 kPSCs 的特点，并比较这些对骨髓间充质干细胞。我们发现 kPSCs，类似于骨髓间充质干细胞，具有免疫抑制能力，并可支持血管丛形成。然而，有组织的细胞类型，作为 kPSCs 表明器官型转录表达签名，包括肾源性转录因子 HoxD10 和 HoxD11 之间的具体区别。kPSCs，与骨髓间充质干细胞，并没有经过肌成纤维细胞转化与 TGF-β 的刺激后，却不能分化为脂肪细胞。此外，kPSCs 加速上皮完整性在肾肾小管上皮伤口划痕实验中，没有观察到与骨髓间充质干细胞的现象。此增强的创面修复介导肝细胞生长因子释放。此外，kPSCs 改善肾损伤的急性肾损伤¹⁵小鼠模型。因此，kPSCs 似乎有优越的肾脏修复能力，是一个有趣的新来源细胞治疗在肾脏疾病。

为了能为细胞治疗目的使用 kPSCs，kPSCs 应隔离在临床分级方式，与临床分级酶和协议。此外，要能够从 1 捐助几个治疗 kPSCs，应该得到足够的单元格数目。在这里我们展示细节，从整个移植级肾，kPSCs 的临床分级分离过程中产生足够数量的细胞用于临床细胞治疗。

Protocol

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.

Representative Results

在图 1中总结出的临床分级 kPSCs 隔离方法。原油肾细胞是从人类移植年级肾脏胶原酶灌注中分离。由此产生的细胞悬浮培养直到 5%血小板裂解液中汇合。然后基于 NG2 表达，血管周围间质细胞分数是孤立的。 kPSCs 塑料贴壁纺锤状细胞 (图 2A)，积极为 CD73 CD90、 CD105、 血管周围的间质标记标记 NG2、 血小板 B、 CD146，而消极 CD31、 CD34、 CD45 和 HLA-DR (图 2B)。通常情况下，kPSCs 均匀人口可达到通道 4 和 kPSCs 到达周围通道 9-10 (图 2C) 衰老。因而，我们建议进行通道 5-8 之间的实验。 要评价功能能力的孤立的 kPSCs，我们请执行体外肾上皮伤口划痕检测对每一批新的 kPSCs，正如我们先前表明的 kPSCs 培养液可以加速上皮伤口愈合在此划伤测定15。KPSC 培养液是通过培养 48 h alphaMEM 5%血小板裂解液中 kPSCs 和收集上清液。接下来，永生化人肾近端小管上皮细胞 (港币 2)17养殖直到汇合，然后划伤的伤口。然后要么 kPSCs 或控制介质条件培养液的中添加到井和伤口愈合的速度衡量。当添加的 kPSCs 培养液时，伤口关闭明显更快 (图 2D)。 图 1： 临床分级分离方法的人类 kPSCs.洗由此产生的细胞悬液移植级肾是空心和经肾动脉 (A-G) 胶原酶灌注和要么冻存或投入文化 (H-K)。细胞达到合流 (L) 后，NG2 细胞富集被执行 (M)。细胞消化时他们要么汇合，已停止增殖或当 3D 结构出现 (N-P)。KPSCs 的发布标准是不育、 标志物表达及增强肾小管上皮伤口愈合 (Q) 的能力。箭头： 肾动脉。条码秤 =请点击这里查看此图的大版本。 200 µ m。 图 2:表征人类 kPSCs。) KPSCs 是纺锤状、 塑料贴壁细胞。B) kPSCs 是为间充质标记 CD73，CD90、 CD105，血管周围标记 NG2、 血小板 β 和 CD146，而消极 CD31、 CD34 和 CD45 阳性。kPSCs 表达 MHC 类我 (HLA-ABC) 但不是类 II (HLA-DR)。C) 生长特性的三个不同的 kPSC 捐助者从流式证实均匀 NG2 积极的人口 （通道 4）。kPSCs 到达周围通道 9-10 的衰老。D) kPSCs 是能够提高肾上皮修复伤口划痕检测中。代表的形象，控制介质及 kPSC 条件介质在 t = 0、 4、 8 和 12 h 显示。条码秤 a） = 200 µ m，d) =请点击这里查看此图的大版本。 100 µ m。

Discussion

血管周围细胞已分离出许多不同的人类固体器官，包括胰腺、 脂肪、 软骨和肾脏^9，，1516。然而，大多数的方法，基于小样本的组织，这是解剖和事后处理消化酶。此外，这通常不会执行与临床分级产品。这使得这些策略不太适合直接临床翻译大量的临床分级细胞是必要。

在这里我们展示整个器官灌注的临床分级酶和材料基于人类 kPSCs 新型分离方法。议定书 》 是改编自目前使用中的临床应用在我们中心¹⁸临床胰岛分离协议。

这是首次临床分级方法可以达到很大数量的 kPSCs。在细胞产率变异性是主要是捐助者的依赖。然而，当我们目前获得的三个不同的捐助者将原油细胞悬浮液的一小部分细胞产率推断，在理论上，可以实现 x 10¹² kPSCs 每捐助 2.7 平均产量。由于 MSC 治疗通常包括用 1-2 x 10⁶细胞/公斤身体重量⁴2 细胞输注，这些单元格数字已足以让同种异体治疗的几个病人。

在分离过程中的一个重要步骤是胶原酶消化的持续时间。当消化期太短，则会继续一大簇的组织将对文化更难。当灌注时间过长时，增加的细胞死亡可能观察到。因此，尽快肾脏开始变得柔软和不透明液体，应轻轻按摩肾脏和胶原酶治疗应停止。

另一个关键的一步后 NG2 细胞富集是细胞的培养。有时后 NG2 细胞富集，不开始血管周细胞增殖或开始在三维结构中成长。在这种情况下，当细胞消化，加赛，细胞将通常开始生长单层培养。

为起始原料，主要用于外科手术的原因被丢弃的人类移植等级肾脏被使用。这些都是无重大肺间质纤维化的功能器官。我们承认，这可能是一个限制，因为这是比较少见，很难获取器官源。移植的肾可能是另一个来源;然而，根据肾取出的原因，这些肾脏可能包含更多的纤维化和因而肌成纤维细胞，并因此要小心因为肌成纤维细胞可能分离、 培养而不是血管周围细胞。

KPSCs 分离与此协议显示有机典型属性与肾上皮伤口愈合能力¹⁵，kPSCs 在肾脏疾病细胞治疗以及移植将是一个有趣的未来应用。为此目的，有的细胞产品交付的几种策略。第一种策略是输液的 kPSCs，作为目前 bmMSC 临床研究中。另一个有趣的应用程序是使用 kPSCs 或 kPSC 排泄因素在移植前机器灌注。这种方式，移植肾的质量可能会提高移植后可能导致肾功能改善。对于这两种策略，kPSCs 是有趣新的细胞来源，进一步探讨用于临床治疗。

Materials

Baxter bags	Fenwal	R4R-7004
Human platelets	Sanquin		hospital surplus material expired for less than 2 days is used
platelet lysate			custom made
Disposable sterile bottles	Corning	09-761-11
500ml PP centrifuge tubes	Corning	431123
a-MEM medium	Lonza	BE12-169F
glutamax	thermo fisher	35050038
pen/strep	Invitrogen	15070063
transfusion system	Codan	455609
DMEM-F12	life technologies	11320-074
normal human serum	Sanquin
Hepes 1M	Lonza	BE-17-737E
NaHCO3 7.5%	Lonza	BE17-613E
CaCl2 1M	Sigma-Aldrich	10043-52-4
UW	Bridge to life	32911
Heparin	Leo Pharma
collagenase NB1 GMP grade	SERVA	17455.03
DMSO	Sigma Aldrich	D2650-100ml
surgical drapes	3M Nederland	DH999969404
perfusion pump	metrohm	x007528300
pump head	metrohm	x077202600
perfusion tray			custom made
LS25 masterflex tubes	Masterflex	HV-96410-25
accessory spike	Gambro DASCO	6038020
pulmozyme	Roche
culture flasks 25 cm2	greiner	690175
culture flask 75 cm2	greiner	658170
culture flask 175 cm2	greiner	661160
Trypsin	sigma	t4174
BSA	Sigma	A2153
EDTA	Sigma-Aldrich	E5134-500g
FcR blocking reagent	miltenyi	130-059-901
anti melanoma beads (NG2)	miltenyi	130-090-452
cellstrainer 70 µm	Corning	352350
LS columns	Miltenyi	130-042-401
MACS magnet	miltenyi	130-090-976
CD34 FITC	BD	555821
CD45 APC	BD	555485
CD146 PE	BD	550315
NG2 APC	R&D	FAB2585A
CD90 PE	BD	555596
HLA ABC APC	BD	555555
CD105 FITC	Ancell	326-040
HLA DR APC	BD	559866
CD56 PE	BD	555516
CD73 PE	BD	550257
CD31 FITC	BD	555445
CD133 PE	miltenyi	130-090-853
PDGF-r	R&D	mab 1263
mouse IgG1 FITC	BD	345815
mouse IgG1 PE	BD	345816
mouse IgG1 APC	BD	345818
IgG2b PE	BD	555743
goat anti mouse PE	Dako	R0480
sodium azide	Merck	822335
DMEM Ham’s F12	Gibco	31331-028
ITS (insul, transferrin, selenium)	Sigma	I1884
hydrocortisone	Sigma	H0135
triiodothyrinine	Sigma	T5516
epidermal growth factor	sigma	E9644
Immortalized human renal PTEC (HK2)			courtesey of M. Ryan, university college Dublin