利用HEK293悬浮细胞生产高产量腺相关病毒载体批次

摘要

本文介绍了一种基于悬浮培养HEK293细胞的腺相关病毒（AAV）生产方案，该方法利用商业供应商提供的可用于研究目的的组分，减少了病毒载体生产所需的时间和劳动投入。

摘要

腺相关病毒载体（AAVs）是研究中枢神经系统（CNS）的有力工具。新型衣壳如 AAV.PHP.eB 可通过尾静脉注射在小鼠中实现 CNS 的广泛转导。为达到相当的转导效率，需使用至少 1 x 10¹¹ 基因拷贝数/小鼠）相比，直接注射到中枢神经系统实质中所需的剂量更低。在我们课题组中，AAV的制备（包括AAV.PHP.eB）依赖于贴壁培养的HEK293T细胞和三质粒共转染法。采用贴壁细胞获得高产量AAV的过程需要大量人力和材料投入。这一限制促使我们开发了一种基于锥形管的悬浮细胞培养方案。我们将贴壁细胞生产的AAV与悬浮培养法生产的AAV进行了比较，并对比了使用聚乙烯亚胺（Polyethylenimine）或TransIt转染试剂进行悬浮培养的效果。AAV载体通过碘克沙醇梯度超速离心法纯化，随后使用离心过滤装置进行缓冲液置换和浓缩。采用贴壁培养方法，我们平均获得了2.6 × 10¹² 总基因组拷贝数（GC），而悬浮法和聚乙烯亚胺（Polyethylenimine）获得的结果为7.7 × 10¹² GC 总量，而 TransIt 产生了 2.4 × 10¹³ GC 总量。两者之间没有差异 体内 与贴壁细胞系统相比，悬浮细胞系统生产的载体的转导效率。综上所述，本文介绍了一种基于悬浮培养HEK293细胞的AAV生产方案，该方案可显著减少载体生产所需的时间和劳动投入，同时使用商业供应商提供的用于科研目的的组分，可获得高出3至9倍的产量。

引言

腺相关病毒（AAV）于1965年被发现，此后已广泛应用于多种研究领域¹。在神经科学研究中，AAV被用于研究基因和神经元功能、绘制神经环路图谱，或构建疾病动物模型²。传统上，这些应用通常通过直接注射到目标部位实现，因为大多数天然血清型无法穿过血脑屏障，或需要高剂量才能穿透^1,2,3。

随着AAV.PHP.B⁴以及下一代衣壳（如AAV.PHP.eB⁵和AAV.CAP-B10⁶）的发现，现已可通过简单的全身性注射靶向中枢神经系统（CNS）。空间定位图谱揭示了AAV.PHP.eB在细胞水平上所靶向的细胞类型^6,7。结合特定的启动子/增强子，这些衣壳为神经科学家提供了广泛的研究机会，使其能够通过无创的AAV递送方式研究基因与脑功能^4,8。

与AAV9（通常为4 × 10¹²基因组拷贝（GC）/小鼠）相比，AAV.PHP.eB所需的剂量较低（通常为1至5 × 10¹¹基因组拷贝（GC）/小鼠）⁷，但相较于直接注射策略（通常为1 × 10⁹ GC/µL注射），仍需制备更多的病毒载体。大多数天然血清型可通过经典的贴壁细胞培养系统结合碘克沙醇纯化方法进行生产^9,10,11,12。然而，对于AAV.PHP.eB而言，该方法需要耗费大量人力进行细胞培养与转染，才能获得一次实验所需的足够载体量⁸。因此，开发了在锥形管中进行悬浮细胞培养以生产AAV的方法。容量可达300 mL的锥形管结构紧凑，可节省培养箱空间和塑料耗材。与在15 cm培养皿上的贴壁细胞相比，悬浮细胞更易于大规模培养和操作。本方案中的转染组分保持不变，因此先前用于贴壁细胞系统的质粒可直接用于基于悬浮细胞的本方案。该方案已成功转移至实验室其他研究人员，并已成功应用于多种衣壳和载体构建体的制备。

方案

All experimental procedures were approved by the institutional animal care and use committee of the Royal Netherlands Academy of Sciences (KNAW) and were in accordance with the Dutch Law on Animal Experimentation under project number AVD8010020199126. In Figure 1, a schematic overview of the complete protocol is provided. From seeding cells to AAV purification, the protocol takes 6 days to complete.

1. Reagent preparation

1. Plasmid purification
   1. Perform plasmid extraction as described by the manufacturer with a few adjustments to ensure sterility of the plasmids.
   2. Prepare 70% ethanol using sterile distilled water and absolute ethanol.
   3. After the isopropanol centrifugation step, dry plasmids in the flow cabinet hood and add sterile 70% ethanol to the tubes. After ethanol precipitation, dry the plasmids in the flow cabinet hood and resuspend the plasmid in sterile distilled water. Take care to use sterilized microcentrifuge tubes.
   4. Take an aliquot for quantification, restriction analysis, and, if needed, sequencing. It is advised to check the integrity of the inverted terminal repeats (ITRs) as mutations can have a negative impact on yield. Adjust the sample concentration to 1 µg/µL.
2. Preparation of 10x Tween lysis buffer
   1. Dissolve 30.3 g Tris buffer and 2.033 g MgCl₂.6H₂O in 400 mL of sterile distilled water, set pH to 8.0, and increase total volume to 450 mL.
   2. Keep Tween 20 sterile, add 50 mL of Tween 20 to buffer solution in the hood, and filter sterilize using a 0.2 µM vacuum filter.
3. Iodixanol dilutions
   1. Iodixanol solution is provided at 60% concentration. Use the starting solution to make 15%, 25%, and 40% solutions.
   2. For 15% solution, dilute 60 mL of 60% solution with 48 mL of 5 M NaCl and 48 mL of PBS-MK (5x PBS with 5 mM MgCl₂ and 12.5 mM KCl), and add distilled water up to 240 mL.
   3. For 25% solution, dilute 67 mL of 60% solution and 32 mL of PBS-MK. Add distilled water up to 160 mL. Mix well and add 1.6 mL of phenol red solution.
   4. For 40% solution, dilute 160 mL of 60% solution with 48 mL of PBS-MK and add distilled water up to 240 mL. For 60% solution, add 1 mL of phenol red to 100 mL of 60% solution.
4. PBS 5% sucrose solution
   1. Add 25 g of sucrose to a 500 mL bottle of DPBS without calcium or magnesium. Shake well and filter using a 0.2 µM bottle vacuum filter.
5. Polyethylene glycol (PEG) 8000 solution
   1. Dissolve 400 g of PEG 8000 and 24 g of NaCl into sterile distilled water and adjust to a final volume of 1000 mL. Stir with heating until fully dissolved. Adjust pH to 7.4 using pH paper.
   2. Filter sterilize using a 0.2 µM bottle vacuum filter to obtain a 40% PEG 8000 solution. Note that filtration will take a while due to the viscosity of the solution-store at 4 ˚C.

2. Culture of HEK293 suspension cells

1. Use wipes soaked in 70% ethanol for cleaning. Clean biosafety hood, prepare and clean all materials needed for culturing cells.
2. Prewarm 30 mL of suspension cell medium to 37 °C in a 50 mL conical culturing tube. Retrieve viral production cells from liquid nitrogen. Quickly thaw cells in a 37 °C water bath. Just before the vial is completely thawed, clean it with a wipe soaked in 70% ethanol and transfer the vial to the biosafety hood.
   NOTE: The details of the viral production cells used here are provided in the Table of Materials.
3. Quickly transfer cells to the prewarmed 50 mL conical culturing tube. Culture cells in a shaking incubator at 37 °C, 80% humidity, 8% CO₂, 200 revolutions per min (RPM), and a shaking diameter of 50 mm. Passage cells, once viability is above 90% and cell density is above 1-3 x 10⁶ cells/mL.
   NOTE: The incubator used has a shaking diameter of 50 mm; culture conditions should be adjusted for a different shaking diameter.
4. Passage cells 3-4 days after thawing. Check culture tube(s) to make sure there are no signs of contamination; for example, fungus will appear as a discolored (black or green) ring.
5. Prepare 400 µL of 0.4% trypan blue solution per sample using a 1 mL stripette.
6. Quickly retrieve suspension cells from the incubator. Immediately extract 500 µL from the tube using a 1 mL stripette. Gently pipet up and down.
7. Add 100 µL of cell suspension to the prepared trypan blue tube. Gently invert the tube to mix; do not pipet to mix, as this will lead to cell death. Return cells to incubator.
8. Take 50 µL of trypan blue-treated cell suspension and apply it to the hemocytometer slide. Count total viable cells and calculate the amount of cell suspension and medium needed for passage or transfection the following day.
9. Warm medium in the incubator for 10-15 min. Add cell suspension to warmed medium and return to incubator. For passaging cells for 3 days (i.e., Monday-Thursday), set to 0.5 x 10⁶ cells/mL; for passaging cells for 4 days (i.e., Thursday-Monday), set to 0.3 x 10⁶ cells/mL.
   NOTE: According to the manufacturer, viral production cells double every 26 h and should be between 3.5 x 10⁶ and 5.5 x 10⁶ before passaging. Cells can be kept for up to passage 20.

3. Transfection

1. Day 1
   1. At 24 h before transfection, culture 1 x 10⁶ cells per/mL in 300 mL of media.
2. Day 2
   1. Warm medium, plasmids, and transfection reagent to room temperature. For the amounts to prepare, see Table 1.
   2. Briefly vortex plasmids and reagents. Add plasmids to the prepared medium, vortex. Add transfection reagent and vortex briefly. Do not agitate the mixture after this step. Incubate at room temperature for 30 min.
   3. In the meantime, count cells prepared on day 1. Cells should be between 2 and 2.5 x 10⁶ cells per mL and > 95% viable.
   4. After incubation, dropwise, add the transfection mixture to cells while gently swirling the tube. Incubate for 72-96 h.

4. Harvesting the cells

1. Day 5
   1. Add 33 mL of 10x cell lysis buffer (500 mM Tris pH 8, 10% Tween 20, 20 mM MgCl₂), mix by gently shaking, and incubate at 37 °C for 1.5 h with shaking.
   2. Centrifuge at 3428 x g at 4 °C for 60 min. Filter through a 0.45 µM PES vacuum filter to clarify the cell lysate, leaving the clarified lysate in the container of the filter.
      NOTE: Optional after filtration: take 50 µL of sample for quantitative-PCR (Q-PCR).
   3. Add 90 mL of 40% PEG 8000 solution to 360 mL of filtered cell lysate.
   4. Clean the stir bar with 70% ethanol and add to the sample. Stir on ice or in the cold room at 300 RPM for 1 h. Incubate at 4 °C without stirring overnight.
      NOTE: Incubation times of 1 h to 72 h have been tested. Longer incubation times have a negative effect on overall protein precipitation.

5. Iodixanol purification

1. Day 6
   1. Transfer the entire PEG precipitated culture volume sample to a clean, large conical tube and centrifuge at 2820 x g and 4°C for 15 min.
   2. Discard the supernatant and resuspend the PEG pellet in 15 mL of DPBS with calcium and magnesium. The pellet is difficult to resuspend; take the time to do this carefully.
   3. Transfer the resuspended part to a clean 50 mL tube. PEG will remain on the sides of the bioreactor tube. Add an extra 10 mL, taking care to gather all the PEG precipitate. Transfer all to a 50 mL container for a total volume of 25-30 mL.
   4. Add 40 µL of DNaseI (10 U/µL). Place in the incubator for 1 h 37°C.
      NOTE: Optional after incubation of DNase: take 50 µL of sample for Q-PCR.
   5. Clean stir bars that were used for PEG precipitation well with chloride solution followed by 70% ethanol. Leave stir bars in 70% ethanol for the next experiment.
   6. Fill a 25 mm x 89 mm polyallomer tube using a glass Pasteur pipette with 15.5 mL of concentrated cell lysate in each tube. After adding cell lysate, exchange the glass Pasteur pipette for a new one.
   7. Add iodixanol solutions from 15%-60%: infuse 9 mL of the 15% iodixanol solution gently beneath the cell lysate. Next, add 5 mL of the 25% and 40% iodixanol solutions, and lastly, add 5 mL of the 60% iodixanol solution.
   8. Top off the tube using a syringe with DPBS +/+ to remove most of the air bubbles, taking care not to disturb iodixanol layers. Seal the tube using an electrical tube topper.
   9. Centrifuge in a non-swing rotor for 1 h 10 min at 490,000 x g at 16 °C in an ultracentrifuge. Remove from ultracentrifuge, assemble the tubes in a metal clasp, and prepare collection tubes. Open the rotor in the hood in case of spills during spinning.
   10. Prepare one 50 mL tube (for discarding the rotor tube), one 15 mL tube (for collecting virus), a 5 mL syringe, and a 30G and 19G needle per gradient.
   11. Puncture a hole in the top of the tube with a 30G needle. Leave the needle in place. Place a 19G needle on the syringe.
   12. Carefully puncture the tube just below the 40%/60% interface, which can be seen by the phenol red indicator. Make sure the needle bevel is facing the 40% layer.
   13. Remove the 30G needle from the top of the tube using the non-dominant hand. With the needle beveled up, slowly extract the virus/iodixanol. The aim is to extract between 3 mL and 4.5 mL. About halfway and well into the 40%/clear layer, rotate the needle for the bevel to face down and continue to extract to avoid collection from the protein layer.
   14. Prepare the 50 mL tube with the non-dominant hand, carefully extract the needle while placing the tube in the 50 mL tube and discard. Dilute the AAV/iodixanol suspension 5x in DPBS by filling up to 15 mL.
   15. Transfer to a centrifugal filter tube and centrifuge at 3428 x g, 4 °C for 10 min. Discard flow-through; gently remove the holder of the filter and tip the bottom container into a waste bottle. Add 15 mL of PBS-5% sucrose to filtrate and resuspend.
   16. Centrifuge at 3428 x g at 4 ˚C for 20 min. Repeat buffer exchange at least 3x. Store virus (~150-250 µL) at 4 °C (PHP.B variants a maximum of 3 months) or aliquot into 50 µL aliquots and store at -80 °C for long term.
       NOTE: PHP.B capsid variants are sensitive to freeze/thaw, so this should be avoided.
   17. Perform titration as described in a previous protocol¹⁰.

结果

大多数学术实验室使用贴壁的 HEK293T 细胞进行 AAV 生产^8,9。虽然在需要少量 AAV 直接注射时该方法效果相对较好，但若使用 AAV.PHP.eB 等全身性衣壳实现相似的转导效率，则需要高 100 倍的滴度（至少 1 × 10¹¹ GC/小鼠）。

本方案建立了利用在锥形管中悬浮培养的HEK293细胞生产AAV的方法。小规模培养可在50 mL管中进行，大规模培养则使用600 mL管。针对特定摇床（见材料表），可使用一种支架，最多可同时培养16根大管。该支架需与专用于50 mL管的特殊插件配合使用（图2A）。该系统显著缩短了本方案中培养和转染阶段的操作时间。初始生产实验采用表达荧光素酶和绿色荧光蛋白（GFP）的报告基因质粒进行^10,11。同时，按照先前发表的方案¹²，在12个15 cm²培养板中平行制备GFP载体。使用聚乙烯亚胺（PEI）转染时，平均总产量达到7.7 × 10¹² GC；使用TransIt转染时，总产量为2.4 × 10¹³ GC（图2B）。与贴壁培养获得的滴度（2.6 × 10¹²）相比，PEI转染的产量提高了近3倍，TransIt转染则提高了9.2倍。在转染悬浮细胞时，TransIt的产量比PEI高出约3倍（图2B）。

随后，对AAV载体的性能进行了测试 体内GFP 载体使用 PEI 和 TransIt 制备，并与采用经典贴壁系统制备的 GFP 载体进行比较。注射后 4 周，处死小鼠（6 周龄、体重 20–25 g 的 C57BL/6 雌性小鼠），通过组织学方法评估小鼠脑组织中的 GFP 表达情况图3A）。在脑部矢状切片上进行了比较分析。使用这些制备方法产生的病毒在转导模式上未观察到差异。图3B）。还评估了使用任一方法产生的载体的荧光素酶活性。转导效率通过测定 体内 3周；无论使用哪种转染试剂，随时间变化的表达模式均相似（图4).

接下来，该方案由团队其他成员进行了测试和实施（表2）。使用PEI制备其他衣壳均获得成功，平均产量达到3.5 × 10¹² GC病毒载体。在B10衣壳的制备中，使用TransIt相比PEI可实现3倍的产量提升。在另一项研究中，将多个构建体包装于AAv.PHP.eB中，平均产量为3.7 × 10¹²，足以以每只小鼠5 × 10¹¹ GC的剂量静脉注射7只小鼠。这些结果表明，该方案可被多位研究人员成功应用于不同的衣壳与构建体组合。

图1：生产的示意图概述。 第1天（D1），在300 mL试管中培养细胞。第2天（D2），用相应的质粒转染细胞。第5天（D5），收获细胞并使用10倍Tween裂解缓冲液裂解。裂解后，通过离心去除细胞碎片。随后，将细胞裂解物过滤以去除大分子蛋白，并使用聚乙二醇（PEG）8000沉淀腺相关病毒（AAV）。第6天（D6），进行碘克沙醇梯度纯化和浓缩。将前一天获得的PEG浓缩物用DNase处理，然后进行碘克沙醇梯度离心。纯化后的载体随后使用离心过滤器进行脱盐和浓缩。请点击此处查看该图的放大版本。

图 2：培养箱设置及AAV产量。 （A）病毒生产细胞在具有50 cm振荡直径的摇床培养箱中培养。该培养箱配备由机电部门制造的适用于600 mL管的适配板和适用于50 mL管的特殊插件。（B）在初始设置后，使用聚乙烯亚胺（PEI；总产量7.7 × 10¹² GC，n=5）或TransIt（总产量2.4 × 10¹³ GC，n=5）作为转染方法的报告基因构建体，与Verhaagen等¹²所述的传统贴壁培养方法（总产量2.6 × 10¹² GC，n=5）的产量进行比较。数据以均值±标准差表示。请点击此处查看此图的放大版本。

图3：转染后GFP表达模式 体内 评估 (A典型的AAV载体结构示意图，包含普遍性启动子CMV immediate-early增强子、鸡β-actin启动子和兔β-珠蛋白剪接受体位点（CAG），其后依次为绿色荧光蛋白（GFP）、土拨鼠肝炎病毒转录后调控元件（WHV WPRE）和多聚腺苷酸化尾部（pA）¹³在此，6周龄雌性小鼠（n=3）通过尾静脉注射接受含有5 x 10¹¹ 总基因组拷贝数（gc）/小鼠。注射后4周，处死小鼠，通过组织学方法分析脑组织。B使用贴壁的HEK293T细胞或悬浮细胞，辅以聚乙烯亚胺（PEI）或TransIt转染试剂进行生产时，冠状切面中典型的转导模式如图所示，无论采用何种生产方法，转导模式均相似。比例尺 = 1000 µm。 请点击此处查看此图的放大版本。

图4：不同转染方法下荧光素酶活性相似。（A）所用构建体的示意图，包含普遍性启动子CMV即时早期增强子、鸡β-肌动蛋白启动子和兔β-珠蛋白剪接受体位点（CAG）驱动的荧光素酶（LUC），带有V5标签以及牛生长激素多聚腺苷酸化尾（BGHpA）；每只小鼠经尾静脉注射总量为5 × 10¹¹ GC（n=3），每周检测颅部区域（示例图像中红色圆圈标记区域）的荧光素酶活性，以生物发光图像表示。（B）生物发光活性以相对光单位（RLU）表示，蓝色柱代表PEI制备组，棕色柱代表TransIt组。在所选脑区，各组之间的荧光素酶活性无显著差异。数据以均值±标准差表示。请点击此处查看该图的放大版本。

表1：转染参数。 请点击此处下载该表格。

表2：所采用方案的产量。 其他研究人员用于生产AAV的悬浮培养方案。请点击此处下载该表格。

讨论

系统性给予 AAV 是一种强大的工具 将基因转移至中枢神经系统；然而，腺相关病毒（AAV）的生产过程成本高昂且费时费力。通过使用悬浮细胞，相较于在15 cm培养皿中贴壁培养HEK293T细胞，可减少人力投入和塑料耗材的使用。² 培养板。此外，本实验所采用的锥形管易于操作，并能最大限度地利用实验室空间。该方案由两名研究人员建立，随后被实验室其他人员推广应用。三位独立研究人员进行的一系列生产实验平均每次制备的病毒载体足以注射6至7只小鼠。

此前已有文献描述过HEK293悬浮细胞的使用^13,14。然而，该文献中所述的悬浮细胞系并未对科研用途免费开放，这为学术研究人员应用所述方法带来了瓶颈。本实验方案中描述的悬浮细胞系可免费用于科研目的。悬浮细胞的培养采用锥形管而非锥形瓶，以节省空间和时间。使用锥形瓶最多可同时培养4个反应，而使用锥形管则可同时培养12个反应。锥形管可直接转移至离心机中进行收获。

悬浮细胞培养系统的另一种选择是杆状病毒系统。杆状病毒系统的一个优势在于，这些细胞和培养基均为公开可得，且易于应用。对于学术研究者而言，Addgene 等质粒库提供了大量即用型腺相关病毒（AAV）转移质粒的资源¹²。尽管这些质粒也可能存在一些问题，例如ITR序列缺陷，但它们仍可作为即插即用实验的良好来源。本研究选择保留基于HEK293的生产系统，因为现有的质粒可直接用于生产，无需将构建体转移至杆状病毒中。

在转染过程中，聚乙烯亚胺相对便宜，因此是首选的转染试剂。作为替代方案，评估了一种新型转染方法（TransIt），以提高载体的产量。使用该方法时，仅需一半量的质粒即可实现三倍的产量提升。这使得TransIt在需要制备较大批次载体时成为一种具有吸引力的替代转染试剂。然而，其局限性在于成本较高，因此在小批量制备中吸引力较低。

综上所述，本文介绍了一种可在学术实验室环境中利用悬浮细胞高效生产腺相关病毒（AAV）的实验方案。同时描述了在学术实验室条件下可用于高效生产AAV的工具。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
39 mL，快速密封圆底聚丙烯管，25 x 89 mm - 50个/包	Beckman Coulter	342414
600 mL 锥形管适配器，用于 S-4x1000 转子	eppendorf	5920701002
适配板，适用于 16 个 600 mL 生物反应器	Infors HT/ TPP	587633
防气溶胶盖，用于 750 mL 圆形桶	eppendorf	5820747005
离心机 5920 R G，230 V，50-60 Hz，包含 S-4x1000 转子、圆形桶及 15 mL/50 mL 锥形管适配器	eppendorf	5948000315
蒸馏水	Gibco	15230147
DNase I 重组，RNase-free	Roche	4716728001
DNase I 重组，RNase-free	Roche	4716728001
含钙、镁的 DPBS	Gibco	14040091
无钙、无镁的 DPBS	Gibco	14190144
Fisherbrand 一次性 PES 滤器单元 0.2 µm	Fisher	FB12566504
Fisherbrand 一次性 PES 滤器单元 0.45 µm	Fisher	FB12566505
50 mL 培养管支架，也适用于 Falcon 管	Infors HT/ TPP	31362
600 mL 细胞培养管支架	Infors HT/ TPP	66129
培养箱 Minitron 50 mm	Infors HT	500043
LV-MAX 生产培养基	Gibco	A3583401
N-Tray 通用型	Infors HT/ TPP	31321
OptiPrep - 碘克沙醇	Serumwerk bernburg	1893
PEI MAX - 转染级线性聚乙烯亚胺盐酸盐（分子量 40,000）	Poly-sciences	24765-100
酚红溶液	Sigma-Aldrich	72420100
聚乙二醇 8000	Sigma-Aldrich	89510
TransIT-VirusGEN	Mirus	Mir 6706
台盼蓝溶液，0.4%	Gibco	5250061
TubeSpin 生物反应器-50 mL	TTP	87050
TubeSpin 生物反应器-600 mL	TTP	87600
病毒生产细胞	Gibco	A35347
Vivaspin 20 超滤管，截留分子量 100,000	Cytvia	28932363