肝-皮肤在芯片上共培养的方法

The Multi-Organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

肝-皮肤在芯片上共培养的方法

 

Introduction

目前用于药物开发的单层或悬浮细胞培养方法无法模仿人类细胞微环境，因此，导致原代人类细胞培养中的快速脱分化和功能丧失。在进行临床试验之前，需要建立具有较高生理相关性的组织模型来预测化合物的有效性和安全性。近年来，标准的体外细胞培养技术已从二维单层培养向三维多细胞模型发展，旨在模拟体内组织微环境。这些系统已经在更准确地预测化合物的作用模式方面显示出了明显的进步。此外，使体外培养条件适应细胞高度特殊化的需要是特别有价值的。

在标准的体外条件下，许多重要的培养参数，如营养物质和氧气的供应、积累代谢产物的去除，以及作用于细胞的机械力，在大多数情况下往往不能完全受控制。许多器官具有生理相关的物质和溶解氧的浓度梯度。然而，这些高度调控和优化的条件与体外条件下组织周围不可控的扩散梯度明显相反，导致环境高度不稳定，限制了细胞的发育。因此，需要更稳定、更可量化的体外条件来保持细胞存活和在较长时间内分化。灌注系统中，培养基成分不断被移除和替换，通常比直接围绕组织的静态培养更好地表征和可控。在静态条件下，细胞分泌物和培养基营养物质的扩散梯度可能会围绕培养细胞。在组织周围引入表征良好的介质流速，使细胞分泌物通过灌注与营养物混合。保证了在整个试验期间确定的细胞微环境，确保稳定的细胞表型和代谢酶的表达。

基于多器官芯片(MOC)的系统最近的发展，兼顾了组织周围受控介质流动、微尺度生物反应器对介质和细胞质量的要求，使得测试过程中所需的待测物质用量减少。到目前为止，已经描述了几种用于组织培养的微流控系统。这些系统中的组织和微流道尺寸比在模拟生理相关的细胞交流中起着特别重要的作用。但由于技术上的限制，如果使用外部泵和培养基储备池，大多数系统的整体循环介质体积相对组织体积过大。Shuler等人的研究小组是第一个开发出一种系统，以确保物质在细胞培养室接触的时间，以及体内相关的组织和微流道尺寸比。这是通过将外部储层缩小到96孔板（也代表“其他组织”隔间）来实现的。为了使我们的MOC平台内的循环介质体积最小化，我们集成了一个芯片上的微蠕动泵，模拟了外部培养基循环流动。这种微型泵能够在可选择的介质流速和剪切应力速率下操作系统。宽度为500 μm高度为100 μm的微流控通道系统连接两个标准化的组织培养空间，每个空间具有96孔板的单个孔的大小。秉承行业标准孔板的尺寸，兼容整合现有的transwell的组织模型。此外，transwell细胞insert小室的垂直位置是可调节的，使组织模型的培养不仅可以直接接触，而且可以被提起并脱离液体。同样，使用该系统也可以实现气-液界面培养。

MOC平台由2 mm高的聚二甲基硅氧烷（PDMS）层和面积为75 x 25 mm²的玻璃载玻片制成，通过低压等离子体氧化永久结合，形成流体密封的微流控流路。PDMS层包含各自的通道和细胞培养室是通过标准软光刻和复制模塑生产的。微流控设计本研究中使用的MOC由每个芯片上两个独立的微流控流路组成，每个流路包含两个相互连接的细胞培养室高100 μm的通道。这允许使用一个多器官芯片进行两个单独的二联组织共培养。泵频率可以调节培养基流速为40 μl/min。

这种二联组织MOC设计使得我们可以在生理流动条件下的串联微流控流路中，在不同的培养空间共培养肝脏球体和皮肤穿刺活检组织。分化的人肝癌细胞（HepaRG）与人肝星状细胞（HHSteC）以24:1的比例共培养形成均匀的肝球体。正如先前的实验所观察到的，这个比率是最佳（使用的肝细胞数量几乎是体内情况的两倍）。皮肤是在气液培养基上培养的transwell细胞insert小室中培养的，从而保证局部接触待测物。这些组织模型共培养28天。此外，芯片的微流控流路被人真皮微血管内皮细胞（HDMEC）完全覆盖，更接近模拟血管系统。

 

Protocol

NOTE：人体少年包皮（Human juvenile prepuce）是在常规包皮环切后进行儿科手术，经知情同意和伦理批准（伦理委员会Charité University Medicine, Berlin, Germany)），并符合相关法律的情况下获得的。

1. Production of Tissue Equivalents for Cultivation in the MOC

1. Aggregate HepaRG and HHSteC in hanging drop plates to generate liver spheroids.

1. In order to achieve the trypsinization of HepaRG cells grown in cell culture flasks (75 cm2), remove the medium from confluent, differentiated monolayer cultures, wash with PBS twice, and add 3 ml of 0.05% Trypsin/EDTA. Incubate for 3 to 5 min at 37 °C and stop the reaction by adding 6 ml of trypsin inhibitor.

2. Centrifuge HepaRG cells at 150 x g for 5 min, remove supernatant, resuspend the cell pellet in 1 ml of HepaRG cell culture medium, and count cells. Cell viability should be >90%.

3. In order to achieve the trypsinization of HHSteC cells, remove the medium from monolayer cultures, wash with PBS twice, and add 3 ml of 0.05% Trypsin/EDTA. Incubate for 5 min at 37 °C and stop the reaction by adding 6 ml of trypsin inhibitor.

4. Centrifuge the HHSteC cells at 150 x g for 5 min, remove supernatant, resuspend the cell pellet in 1 ml of HepaRG cell culture medium, and count cells.

5. Combine HepaRG and HHSteC at a ratio of 24:1 in HepaRG cell culture medium. To do so, adjust cell numbers by diluting cell suspensions in HepaRG cell culture medium and add 1 x 105 cells/ml HHSteC to 4.8 x 106 cells/ml HepaRG cells. Mix carefully.

6. Prepare a hanging drop plate by adding 2 ml of PBS to the hanging drop and receiver plate.

7. Pipette 20 μl of the cell suspension into each well of the hanging drop plate. Always prepare about 10% more hanging drops than you need to retrieve aggregates, as some aggregates are lost during the procedure. Carefully place the plate in a 37 °C incubator. Wait 48 hr for the spheroids to form.

8. In order to retrieve the spheroids, be sure to use pipette tips with wide tip endings or cut the tips of 1 ml pipette tips with a sterile knife in order to widen the opening to about 2 to 3 mm. Use these pipette tips to handle the spheroids without disrupting them.

9. Wash off spheroids carefully from the hanging drop plate by repeatedly adding 1 ml of media to the top of the wells of the hanging drop plate using a pipette. Wash the plate until all the spheroids have fallen off. Spheroids are slightly disc-shaped with a medium diameter of 300 to 400 μm and a height of 200 to 300 μm at this point.

10. Collect the spheroids in the receiver plate and transfer them to 24-well ultra-low attachment plates with a maximum of 20 spheroids per well using the prepared pipette tips. Adjust the media volumes in each well to 0.5 ml. Use 20 aggregates to inoculate one MOC circuit to obtain a miniaturization rate of 1/100,000 regarding in vivo cell numbers.

11. Incubate the spheroids at 37 °C and 5% CO2 until further use in the MOC. Do not store the spheroids longer than three days before usage to assure comparability. Cultivate the aggregates for at least one day in ultra-low attachment plates to retrieve the homogenous spheroids.

2. Pursue either of two approaches to generate skin tissue equivalents: the use of punch biopsies (1.2.1) or the use of ready-made in vitro tissue models (1.3.1).

1. Cut transwells with an incandescent knife below the bracket to prepare 96-well cell culture inserts and store under sterile conditions until further use.

2. Sterilize prepuce samples in 80% ethanol for 30 sec and cut the ring open. Samples should have an average height of 2 mm.

3. Use a biopsy punch to cut biopsies of 4.5 mm diameter to obtain an equal miniaturization ratio for both liver and skin. Load the biopsies into the prepared 96-well transwell inserts with forceps. Take care to position the biopsies with the epidermal side facing upwards.

4. Place cell culture inserts with biopsies in a receiver plate containing HepaRG cell culture medium and store at 37 °C and 5% CO2 until further use in the MOC. Do not store samples longer than 2 to 3 hr.

3. Integrate ready-made in vitro skin models, purchased from various suppliers, into the MOC, making sure that they are in 96-well transwell format. Use the cell culture medium supplied by the vendor or, if a coculture with another tissue is envisaged in a further step, use a minimal medium supporting both tissues. Test the respective minimal medium in prior static experiments for its capacity to support the tissues.

1. Retrieve skin models from the holder plate and cut the 96-well inserts below the bracket with an incandescent knife.

2. Place the inserts back into the receiver plate and store at 37 °C and 5% CO2 until further use in the MOC. Do not store samples longer than one day.

2. MOC Fabrication

1. Mix the PDMS and curing agent at a ratio of 10:1 (v/v) and place the mixture under vacuum for 15 min to remove air bubbles.

2. Meanwhile, treat a polycarbonate cover-plate with the silicon rubber additive at 80 °C for 20 min.

3. Insert Teflon screws into respective holes of the cover-plate to create the four PDMS-free cell culture compartments and the six PDMS

membranes, 500 μm thick, of the micropump.

4. Plug the prepared cover-plate to the master mold of the two microvascular circuits and inject the degassed PDMS. Take care not to integrate

air bubbles into the system. If bubbles emerge, try to remove them by tilting the device.

5. Incubate the system at 80 °C for 60 min to cure the PDMS layer.

6. Remove the master mold and the Teflon screws from the device and bond the PDMS layer to a glass slide with a 75 x 25 mm2 footprint using

low pressure plasma oxidation.

7. Screw special thread MOC adapters to all four cell culture compartments of the cover-plate.

8. Connect syringes containing culture medium to female Luer x ¼-28 male adapters and screw them to the MOC adapters of the cover-plate.

9. Inject the medium into the microfluidic circuit by repeatedly pushing down and pulling up the syringes plungers.

10. Check the proper filling of the channels with medium under the microscope.

3. Endothelialization of the MOC

1. Prior to endothelializing the MOC, flush each MOC circuit with endothelial cell growth medium and incubate it statically for three days at 37 °C and 5% CO2.

1. Sterilize the MOCs using an ethanol wipe and place them under a laminar flow bench. In addition, sterilize two pairs of forceps and two

hexagonal keys for further use.

2. Loosen the caps of the tissue culture compartment of the MOC using the hexagonal keys and remove the caps using the forceps. After inserting the medium, screw caps back on to the MOCs in the same way.

2. In order to achieve the trypsinization of human dermal microvascular endothelial cells (HDMEC), remove the medium from the monolayer cultures, wash with PBS twice, and add 3 ml of 0.05% Trypsin/EDTA. Incubate for 5 min at 37 °C and stop the reaction by adding 6 ml of trypsin inhibitor.

3. Centrifuge the HDMEC at 220 x g for 5 min, remove supernatant, resuspend the cell pellet in 1 ml of endothelial cell growth medium, and count cells. Cell viability should be >90%.

4. Adjust the cell count in the cell suspension to a final concentration of 2 x 107 cells/ml by diluting it with endothelial cell growth medium and transfer 250 μl of it to a 1 ml syringe. Apply this concentration of cells to the MOC to keep the miniaturization rate of 1/100,000 for all organs.

5. Connect the syringe to a female Luer x ¼-28 male adapter, expel the air out of this fitting, and screw it to a special thread MOC adapter. Connect the adapter to one of the two compartments of each MOC circuit.

6. Connect an empty syringe in the same way to the second compartment of the MOC circuit.

7. Inject the cells evenly by pushing down and pulling up the two syringe plungers several times in a consecutive way. Control the infusion of cells under the microscope.

8. Incubate the MOC at 37 °C and 5% CO2 under static conditions for 3 hr to allow the cells to adhere to the channel walls.

9. Remove the chip from the incubator, place it under a laminar flow bench, and replace the syringes and the MOC adapters with special thread MOC cell culture chambers.

10. Add 400 μl of fresh medium to one compartment of each MOC circuit and let it flush through the channels by hydrostatic pressure.

Afterwards, replace the medium in both compartments with 300 μl of fresh medium. 11. Close the compartments using caps, as described in 3.1.2.

12. Connect the chip to the pump control unit. Adjust the pumping velocity to a frequency of 0.475 Hz and cultivate the chip at 37 °C and 5% CO2.

13. Replace the medium of each MOC compartment every one to two days and monitor the cell morphology by light microscopy.

4. Loading of the Chip

1. Place the MOC under the laminar flow bench and open it, as described in step 3.1.2.

2. Remove the media from the tissue culture compartments and replace it with 300 μl of fresh HepaRG cell culture medium.

3. Transfer 20 preformed spheroids to one tissue culture compartment of each MOC circuit using the pipette tips with a wide opening (see steps

1.1.8/9). Close the cap using forceps and hexagonal keys.

4. Transfer 96-well cell culture inserts containing skin equivalents to the remaining tissue culture compartment of each MOC circuit using

forceps. Take care to avoid bubble formation below the membrane of the skin equivalent. In order to do this, insert transwells at a slightly

tilted angle and push down gently. Remove excess medium around the transwell being pushed up from below with a pipette.

5. Close the cap using forceps and hexagonal keys.

5. Connecting the Chip to the Pump Control Unit

1. Set operational parameters in the control units to the values desired. Modify air pressure from 0 to 8,000 mbar, vacuum from 0 to -800 mbar,

and pumping frequency from 0.24 to 2.4 Hz. Set the pumping direction clockwise or counterclockwise.

2. Remove the MOC containing tissue equivalents from under the laminar flow bench and connect it to the pump control units.

3. Following the numeration on the tubes, insert air pressure tubing to the respective fittings on the MOC.

4. Cultivate the MOC at 37 °C and 5% CO2 in an incubator or, in the case of live tissue imaging, use the MOC support to heat the chip to 37 °C and cultivate the chip outside the incubator. Use the heated support to cultivate cells in the MOC under a standard microscope.

6. Performing Media Exchanges, Sampling Media and Exposure to Substances

1. Perform routine media exchange every day or every other day, considering the type of tissue cultured and the metabolic activity of the cells.

1. Retrieve the MOC from the incubator and observe it under the microscope to control media flow rates and check for contamination.

2. Disconnect the MOC from the pump control unit by unplugging the air pressure tubing. Sterilize the MOC using ethanol wipes and put it under the laminar flow bench.

3. Open the tissue culture compartment containing the liver spheroids, as described in step 3.1.2.

4. Remove up to 200 μl from the compartment using a pipette without disrupting the spheroids and store the medium in an empty well of a deep-well plate. Analyze the media sample directly or close the deep-well plate and store the media samples at -80 °C for further analysis.

5. Replace the medium of the MOC with up to 250 μl fresh cell culture medium and close the cap. The difference in the amount of medium removed and replaced accounts for the loss due to small amounts leaking out of the chip when closing the system.

6. Open the cap of the tissue culture compartment holding the skin equivalents at this point to check for tissue intactness. Take care not to introduce air bubbles into the system. Close the cap.

7. Connect the tubing of the pump control unit to the MOC, according to step 5.3, and place the MOC in an incubator.

7. Analyze Daily Media Samples and Perform On-line Analysis

1. Analyze tissue culture performance on-line using live cell imaging or offline, by analyzing the daily media samples. Perform the latter by standard routine enzymatic assays (e.g. lactate dehydrogenase (LDH) activity) or ELISA (e.g. albumin concentration). An online analysis is described in the following.

2. Remove the media of the endothelialized MOC, as described in steps 6.1.1 to 6.1.4, and replace it in both tissue culture compartments with 200 μl of 10 μg/ml fluorophore conjugated acetylated low-density lipoprotein (LDL) solution (diluted in cell culture media).

3. Close the caps. Connect the MOC to the pump control unit, according to step 5.3, and pump it for 30 min at 0.475 Hz to distribute the solution evenly within the microfluidic circuit.

4. Stop the pumping and incubate the MOC statically for 3.5 hr at 37 °C and 5% CO2.

5. Similar to step 7.2, remove the acetylated LDL solution from both compartments and replace it with 400 μl of fresh medium in one of the two compartments.

6. Wait for 3 to 5 min to let the hydrostatic pressure drive the medium through the microfluidic channel circuit.

7. Replace the medium which has flowed through with 300 μl of fresh medium and also fill the second tissue culture compartment with 300 μl of fresh medium.

8. Close the MOC, according to step 3.1.2. Put it under a fluorescence microscope and observe cell growth and viability.

9. Place the stained MOC back in the incubator to continue cultivation. Remove the stain leaking out of the cells with each following media replacement.

8. Retrieve Tissue Equivalents from the MOC and Perform End-point Analyses

1. Retrieve tissue equivalents from the MOC at the end of the experiment for endpoint analyses.

1. In order to retrieve the liver and skin equivalents from the MOC, remove the media from each tissue culture compartment, as described in steps 6.1.1 to 6.1.4.

2. Remove the 96-well cell culture inserts containing the skin from the MOC using forceps. Peel off the membrane carefully from the insert by gripping it on one side with forceps and pulling it down. Take care not to lose the skin equivalent at this point.

3. Freeze the membrane holding the skin equivalent in cryo-embedding compound and store it at -80 °C until further analysis.

2. Remove the liver equivalents similarly from the tissue culture compartment by pipetting them using cut pipette tips (see step 1.1.8/9).

1. Embed the liver spheroids in cryo-embedding compound. Take care not to transfer too much liquid and remove any excess fluid with a pipette. After placing the spheroids onto the embedding compound and removing the medium, add further cryo compound onto the top of the spheroids to fully enclose them.

2. Freeze the liver equivalents and store it at -80 °C until further analysis.

3. Perform endpoint analysis by cutting the tissue equivalents in a cryo-microtome to 8 μm sections and staining for tissue-specific markers, as described in previous protocols.

Representative Results

标准的体外组织培养是在静态条件下进行的，限制了氧气和营养物质向组织的扩散。微流道体系改进的特性，但是经常限于尺寸要求，没有合适的培养基-组织比例。因此，代谢产物被稀释，细胞不能适应环境。本研究中提出的MOC通过微流控通道系统连接两个独立的组织培养室，每个室大小为标准96孔板的单孔大小。小规模的系统和集成的芯片上的微流泵允许培养基体积只有200到800 μl的情况下工作。这对应于肝脏和皮肤组织共培养（总组织体积约26 μl）的总培养基与组织的比例分别为8:1 ~ 31:1。体重73公斤的男子的总的细胞外液的体体积为14.6 L（其中毛细血管间液体体积为5.1 L），体积大约为60L，生理细胞外液体与组织的体积比例为1：4（14.6：60）。因此，相对于生理状态，MOC中整个循环系统的培养基体积仍然较大，但是它代表了迄今为止多器官系统报道中最小的培养基与组织比例。由于保留了行业标准的组织培养格式，研究人员能够将现有的和已经验证过的静态组织模型结合在一个共同的液体流道中。图1显示了可能的MOC单组织或多组织共培养的实验设置示意图。原代组织切片和从细胞系或原代细胞在体外生成的组织等效物可以使用96孔细胞培养小室insert或直接将它们放入组织培养室进行培养。由于连接细胞培养室的通道系统只有100 μm高，超过这些尺寸的组织当量将被保存在培养室中。用HDMECs将MOC流路内皮化，通过提供一种生物血管结构，使其向更接近生理的培养条件迈进了一步。

 

图1:MOC培养的示意图。在标准的体外条件下制备组织接种到MOC中，在动态条件下作为单一培养物或共培养物培养。每天进行培养基取样和端点分析。微流泵的气压通过三根从上面连接到MOC的蓝色管子施加。

按照内皮化方案，动态培养4天内可获得微流控通道电路的融合HDMEC覆盖，如图2所示。细胞很容易粘附在MOC通道壁上，形成一个融合的单层膜，并沿着剪切应力拉长(图2B)。此外，如前所述，细胞覆盖了通道的整个周长。培养4天后直至培养结束，内皮形态未见进一步改变。

 

Discussion

这里描述的MOC平台是一个稳定和强大的工具，用于培养各种来源的组织，在动态介质流动条件下，持续培养。本例中，该平台用于培养原代细胞(HDMEC)，即组织由细胞系(肝聚集物)产生，并与组织活检共培养。在混合培养基中，MOC能够维持三组织共培养长达28天。据作者所知，这是第一次多组织共培养包括活组织，原代细胞和细胞系已经进行了四周的培养。

微流控系统的一个主要缺点是小分子与流体电路表面材料的亲和力。由于微流控系统的表面体积比特别高，这种影响变得更加明显。这里介绍的通道稳定的HDMEC覆盖，可能作为一个生物屏障，阻止分子粘附到MOC上。此外，它还可以作为血管（血液相容性）进行全血循环，防止血液凝结。然而，使用全血作为培养基替代品还不可行，因为完全血管化的类器官还没有实现。目前在体外生成血管化类器官方面的工作已经取得进展，并为下一步的研究指明了方向。

众所周知，在静态二维体外培养条件下，肝细胞随着时间的推移往往会失去其肝脏特异性功能。如果要研究某一药物的代谢，细胞色素P450家族等代谢酶尤为重要。细胞色素P450 3A4是一种与许多外来生物的生物转化有关的酶，细胞色素P450 7A参与胆汁酸合成。在MOC培养超过14天的肝聚集物中一直有表达，表明培养物良好的代谢活性，可用于药物代谢研究。与静态培养相比，MOC中聚集体产生白蛋白的速度是培养条件合适的另一指标。在本研究中观察到的白蛋白产生率与之前报道的微流控芯片（包括HepG2细胞）的值相当，甚至更高，但是数值没有达到原代的人肝细胞培养的值。此外，MOC系统的布局，不允许单独分离胆汁。细胞MRP-2染色显示，聚集物极化形成胆汁小管样结构。然而，这些小管并没有连接通道收集胆汁。这种胆汁与血液腔室的非生理混合必须在将来系统的重新设计之中加以解决。

流动特性的控制是非常重要的，特别是对剪切应力敏感的组织，如肝脏。可以降低泵的压力，降低系统中的峰值剪应力值。其次，这些组织可以嵌入到细胞外基质层或在transwell培养小室insert中培养。后者用多孔膜保护组织不受流路的影响。这些在开始MOC实验之前，需要对每个器官进行相应的调整。在搏动手术中例如，在2.4赫兹相当于比较高的、生理的心脏活动，144次/分钟，测量的剪应力在微流路的通道内达到约25 dyn/cm²。这对应微血管系统的末端较高的生理剪应力相，很好地适用于血管内皮化的实验。然而，由于目前MOC系统的微流控布局只包含一个连接两个器官培养室的液体流路，整个系统使用同一个泵流速度和剪应力速率。因此，要对每个器官的特性进行精确的流速调整并能实现。