? TROUBLESHOOTING  ## 故障排除

x From each plate, pick two or three colonies to check for the correct insertion of sgRNA. Use a sterile pipette tip to inoculate a single colony into a 3-ml culture of LB medium with $100\mu \mathrm{g}{\mathrm{ml}}^{-1}$$100\mu \mathrm{g}{\mathrm{ml}}^{-1}$100 mugml^(-1)100 \mu \mathrm{g} \mathrm{ml}^{-1} ampicillin. Incubate the culture and shake it at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} overnight.
从每个平板中挑选两个或三个菌落以检查 sgRNA 是否正确插入。 使用无菌移液器吸头将单个菌落接种到含有 $100\mu \mathrm{g}{\mathrm{ml}}^{-1}$$100\mu \mathrm{g}{\mathrm{ml}}^{-1}$100 mugml^(-1)100 \mu \mathrm{g} \mathrm{ml}^{-1} 氨苄青霉素的 3 毫升 LB 培养基中。将培养物在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 培养并振摇过夜。
xi Day 3: isolate the plasmid DNA from cultures by using a QIAprep spin miniprep kit according to the manufacturer’s instructions.
xi Day 3：根据制造商的说明，使用 QIAprep 旋转柱 Mini 裂解试剂盒从培养物中分离质粒 DNA。
xii Sequence validation of CRISPR plasmid. Verify the sequence of each colony by sequencing from the U6 promoter using the U6-Fwd primer. Optional: sequence the Cas9 gene by using the Cbh-Fwd and SXRP002-007 primers listed in supplementary Data 1. Reference the sequencing results against the $\mathrm{pSpCas}9(\mathrm{BB})$$\mathrm{pSpCas}9(\mathrm{BB})$pSpCas9(BB)\mathrm{pSpCas} 9(\mathrm{BB}) cloning vector sequence to check that the 20 -nt guide sequence is inserted between the U6 promoter and the remainder of the sgRNA scaffold (Fig. 4c). Details and sequence of the pSpCas 9 (BB) map in GenBank vector map format (*.gb file) can be found at http://crispr.genome-engineering.org/.
## 序列验证 CRISPR 质粒。使用 U6-Fwd 引物从 U6 启动子测序，验证每个菌落的序列。可选：使用补充数据 1 中列出的 Cbh-Fwd 和 SXRP002-007 引物对 Cas9 基因进行测序。参考测序结果与 $\mathrm{pSpCas}9(\mathrm{BB})$$\mathrm{pSpCas}9(\mathrm{BB})$pSpCas9(BB)\mathrm{pSpCas} 9(\mathrm{BB}) 克隆载体序列进行比对，以检查 20-nt 导向序列是否插入 U6 启动子和 sgRNA 支架的其余部分之间（图 4c）。有关 pSpCas9 (BB) 序列图以及 GenBank 载体图格式 (*.gb 文件) 的详细信息，请访问 http://crispr.genome-engineering.org/。
? TROUBLESHOOTING ? 故障排除

■ CRITICAL The CRISPR-Cas system has been used in a number of mammalian cell lines. Conditions may vary for each cell line. Below we detail transfection conditions for HEK 293FT cells. Note that ssODN-mediated HDR transfections, performed with Amaxa SF cell line Nucleofector kit, are described in Steps 14-29. For hESC (HUES9) culturing and transfection, follow Steps 30-53.
## ■ 重要 CRISPR-Cas 系统已被用于多种哺乳动物细胞系。每个细胞系的条件可能不同。以下我们详细介绍 HEK 293FT 细胞的转染条件。请注意，使用 Amaxa SF 细胞系 Nucleofector 试剂盒进行的 ssODN 介导的 HDR 转染在步骤 14-29 中进行描述。对于 hESC (HUES9) 培养和转染，请按照步骤 30-53 进行。
6 HEK 293FT maintenance. Cells are maintained according to the manufacturer’s recommendations. Cells are cultured in D10 medium supplemented with $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% (vol/vol) FBS at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} and $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2}.
6 HEK 293FT 维护。 细胞根据制造商的建议进行维护。 细胞在添加了 $10\mathrm{\%}$$10\mathrm{\%}$10%10 \% （体积/体积）FBS 的 D10 培养基中，在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 和 $5\mathrm{\%}{\mathrm{CO}}_2$$5\mathrm{\%}{\mathrm{CO}}_2$5%CO_(2)5 \% \mathrm{CO}_{2} 下培养。
7 To passage, remove the medium and rinse the cells once by gently adding DPBS to the side of the vessel, so as not to dislodge the cells. Add 2 ml of TrypLE to a T75 flask, and incubate the mixture for 5 min at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C}. Add 10 ml of warm D10 medium to inactivate the trypsin, and transfer the cells to a 50-ml Falcon tube. Dissociate the cells by pipetting them up and down gently, and then reseed them into new flasks as necessary.
在操作 7 时，移除培养基并用 DPBS 对细胞进行一次轻轻的冲洗，以避免细胞移位。向 T75 培养瓶中添加 2 ml 的 TrypLE，然后在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 条件下孵育 5 分钟。加入 10 ml 的温热的 D10 培养基以灭活胰蛋白酶，并将细胞转移到 50 毫升的 Falcon 离心管中。通过上下轻轻吹打来解离细胞，然后根据需要将细胞重新接种到新的培养瓶中。

A CRITICAL STEP We typically passage cells every $2-3$$2-3$2-32-3 d at a split ratio of 1:4 or 1:8, never allowing cells to reach more than $70\mathrm{\%}$$70\mathrm{\%}$70%70 \% confluency. Cells are discarded upon reaching passage number 15 .
关键步骤 我们通常每隔 $2-3$$2-3$2-32-3 d 对细胞进行传代，分裂比例为 1:4 或 1:8，决不允许细胞达到高于 $70\mathrm{\%}$$70\mathrm{\%}$70%70 \% 的 confluent。细胞在达到传代次数 15 时予以丢弃。
8 Preparation of cells for transfection. Plate the well-dissociated cells onto 24well plates in D10 medium without antibiotics 16-24 h before transfection. Seed the cells at a density of $1.3\times {10}^5$$1.3\times {10}^5$1.3 xx10^(5)1.3 \times 10^{5} cells per well in a total volume of $500\mu \mathrm{l}$$500\mu \mathrm{l}$500 mul500 \mu \mathrm{l}. Scale up or down according to the cell line supplier’s manual as needed.
### 8 细胞转染的准备工作。将完全解离的细胞在转染前 16-24 小时内在没有抗生素的 D10 培养基中接种到 24 孔板中。每个孔接种 $1.3\times {10}^5$$1.3\times {10}^5$1.3 xx10^(5)1.3 \times 10^{5} 个细胞，总培养基体积为 $500\mu \mathrm{l}$$500\mu \mathrm{l}$500 mul500 \mu \mathrm{l} 。根据细胞株供应商手册的需要进行放大或缩小。

A CRITICAL STEP Do not plate more cells than the recommended density, as doing so may reduce transfection efficiency.
重要步骤 不要比推荐密度多接种细胞，因为这样做可能会降低转染效率。
9 On the day of transfection, cells are optimal at 70-90% confluency. Cells can be transfected with Lipofectamine 2000 or the Amaxa SF cell line 4D-Nucleofector X kit according to the manufacturers’ instructions. Transfections should be performed as follows: for sgRNAs cloned into pSpCas9(BB), transfect 500 ng of sequence-verified CRISPR plasmid (pSpCas9(sgRNA)); if you are transfecting more than one plasmid (Box 2), mix them at equimolar ratios and use no more than 500 ng of total DNA. For sgRNA amplified by PCR, mix the following:
细胞在转染当天处于 70-90% 的汇合度最佳。根据制造商的说明，可以使用 Lipofectamine 2000 或 Amaxa SF 细胞系 4D-Nucleofector X 试剂盒转染细胞。转染应按以下步骤进行：对于克隆到 pSpCas9(BB) 中的 sgRNA，转染 500 ng 序列验证的 CRISPR 质粒 (pSpCas9(sgRNA))；如果要转染多个质粒（方框 2），则以等摩尔比例混合它们，并使用不超过 500 ng 的总 DNA。对于通过 PCR 扩增的 sgRNA，混合以下物质：

CRITICAL STEP We recommend transfecting in technical triplicates for reliable quantification, and including transfection controls (e.g., GFP plasmid) to monitor transfection efficiency. pSpCas9(sgRNA)-2A-GFP or pSpCas9(sgRNA)-2A-Puro may be used in place of pSpCas9 if fluorescence sorting or drug selection, respectively, is desired. In addition, the pSpCas9(BB) cloning plasmid and/or the sgRNA amplicon may be transfected alone as a negative control for downstream functional assays.
## 关键步骤 建议进行三个生物重复的技术性转染以获得可靠的定量，并包括转染对照（例如，GFP 质粒）以监测转染效率。如果需要进行荧光分选或药物选择，可以使用 pSpCas9(sgRNA)-2A-GFP 或 pSpCas9(sgRNA)-2A-Puro 代替 pSpCas9。此外，pSpCas9(BB) 克隆质粒和/或 sgRNA 扩增子可与下游功能分析一起单独转染作为阴性对照。
10 Add Lipofectamine complex to the cells gently, as HEK 293FT cells can detach easily from the plate, which will result in a lower transfection efficiency.
将 Lipofectamine 复合物轻轻添加到细胞中，因为 HEK 293FT 细胞很容易从平板中剥落，这会导致较低的转染效率。
11 Check cells after 24 h for transfection efficiency. The percentage of fluorescent cells in the transfection control (e.g., GFP) can be estimated by using a fluorescence microscope. Typically, more than $70\mathrm{\%}$$70\mathrm{\%}$70%70 \% of cells are transfected.
11 在 24 小时后检查细胞的转染效率。使用荧光显微镜可以估计转染对照组（例如 GFP）中荧光细胞的百分比。通常，超过 $70\mathrm{\%}$$70\mathrm{\%}$70%70 \% 的细胞被转染。

12 Supplement the culture medium with an additional $500\mu \mathrm{l}$$500\mu \mathrm{l}$500 mul500 \mu \mathrm{l} of warm D10 medium.
12 向培养基中补充 $500\mu \mathrm{l}$$500\mu \mathrm{l}$500 mul500 \mu \mathrm{l} 温暖的 D10 培养基。

■ CRITICAL STEP Add D10 very slowly to the side of the well, and do not use cold medium, as cells can detach easily. Puromycin selection can be applied at a concentration of $1-3\mathrm{g}{\mathrm{ml}}^{-1}$$1-3\mathrm{g}{\mathrm{ml}}^{-1}$1-3gml^(-1)1-3 \mathrm{~g} \mathrm{ml}^{-1} for HEK 293FT cells (may vary depending on the cell line).
■ 关键步骤 将 D10 非常缓慢地添加到容器壁，请勿使用冷培养基，因为细胞容易脱落。对于 HEK 293FT 细胞，Puromycin 选择浓度可设置为 $1-3\mathrm{g}{\mathrm{ml}}^{-1}$$1-3\mathrm{g}{\mathrm{ml}}^{-1}$1-3gml^(-1)1-3 \mathrm{~g} \mathrm{ml}^{-1} （可能因细胞系而异）。
13 Incubate the cells for a total of 48-72 h after transfection before passaging them for downstream applications or harvesting for indel analysis.
13 转染后，将细胞孵育 48-72 小时，然后进行传代以进行下游应用或收集用于插入缺失分析。

14 Linearize 1-2 $\mu \mathrm{g}$$\mu \mathrm{g}$mug\mu \mathrm{g} of targeting vector if possible by cutting once at a restriction site in the vector backbone near one of the homology arms or at the distal end of either homology arm.
14 尽量通过在靠近同源臂之一的载体骨架中的限制性位点或任一末端进行一次切割来线性化靶向载体 1-2 $\mu \mathrm{g}$$\mu \mathrm{g}$mug\mu \mathrm{g} 中的限制性位点。

Alternatively, if you are using ssODNs, simply resuspend them to a final concentration of 10 $\mu \mathrm{M}$$\mu \mathrm{M}$muM\mu \mathrm{M} (see Step 4) and skip Steps 15 and 16.
或者，如果您使用的是 ssODN，只需将它们重新悬浮到最终浓度为 10 $\mu \mathrm{M}$$\mu \mathrm{M}$muM\mu \mathrm{M} (见步骤 4)，并跳过步骤 15 和 16。
15 Run a small amount of the linearized plasmid alongside uncut plasmid on a 0.8 $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% (wt/vol) agarose gel to check for successful linearization. Linearized plasmids should run above the supercoiled plasmid.
15. 在 0.8 $1\mathrm{\%}$$1\mathrm{\%}$1%1 \% (重量/体积) 琼脂糖凝胶上，与未切割质粒一起运行少量的线性化质粒，以检查线性化是否成功。线性化质粒应该在超螺旋质粒之上运行。
16 Purify the linearized plasmid with the QIAQuick PCR Purification kit, and elute in $35\mu \mathrm{l}$$35\mu \mathrm{l}$35 mul35 \mu \mathrm{l} of EB buffer.
16 用 QIAQuick PCR Purification 试剂盒纯化线性化质粒，并用 $35\mu \mathrm{l}$$35\mu \mathrm{l}$35 mul35 \mu \mathrm{l} EB 缓冲液洗脱。
17 Preparation of cells for transfection. Culture HEK 293FT in T75 or T225 flasks. Plan ahead to have sufficient cells for the day of transfection ( $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} cells per transfection if you are using the Amaxa SF cell line 4D-Nucleofector X kit S).
## 17 细胞转染前准备 将 HEK 293FT 细胞培养在 T75 或 T225 培养瓶中。提前计划好，以便在转染当天获得足够的细胞（如果您使用的是 Amaxa SF 细胞系 4D-Nucleofector X 试剂盒 S，则每次转染需要 $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} 个细胞）。
18 Prewarming plates for transfection. Add 1 ml of warm D10 medium into each well of a 12-well plate. Place the plates in the incubator to keep the medium warm.
18 个预热转染板。在 12 孔板的每个孔中加入 1 毫升温热的 D10 培养基。将板置于培养箱中，保持培养基温热。
19 Use option A in the table below for preparing the co-transfection of the HDR targeting plasmid with the Cas9 plasmid or option B for the co-transfection of ssODN with the Cas9 plasmid. To prepare transfection controls, see Step 9. If an sgRNA is cloned into pSpCas9(BB)-2A-GFP, cells may also be sorted by fluorescence. If you are using Cas9 nickase to mediate HDR, substitute pSpCas9(sgRNA) with pSpCas9n(sgRNA) from Step 5B(v).
19. 使用下表中的选项 A 准备 HDR 靶向质粒与 Cas9 质粒的共转染，或选项 B 准备 ssODN 与 Cas9 质粒的共转染。要制备转染对照，请参见步骤 9。如果将 sgRNA 克隆到 pSpCas9(BB)-2A-GFP 中，也可以通过荧光对细胞进行分选。如果您正在使用 Cas9 切割酶介导 HDR，请用步骤 5B(v) 中的 pSpCas9n(sgRNA) 替换 pSpCas9(sgRNA)。
$\mathrm{△}$$\mathrm{△}$/_\\triangle CRITICAL STEP For HDR applications, we recommend cloning sgRNA guides into one of the sgRNA expression plasmids described in Step 5B, rather than using the PCRbased expression approach.
$\mathrm{△}$$\mathrm{△}$/_\\triangle ##

20 Dissociation of cells for transfection. Remove the medium and rinse the cells once gently with DPBS, taking care not to dislodge cells. Add 2 ml of TrypLE to a T75 flask and incubate it for 5 min at ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C}, and then add 10 ml of warm D10 medium and triturate gently in a 50-ml Falcon tube.
将细胞用于转染前进行解离。除去培养基，并用 DPBS 轻轻冲洗细胞一次，注意不要使细胞脱落。向 T75 培养瓶中添加 2 ml TrypLE，在 ${37}^{\circ }\mathrm{C}$${37}^{\circ }\mathrm{C}$37^(@)C37^{\circ} \mathrm{C} 下孵育 5 分钟，然后添加 10 ml 温热的 D10 培养基，并在 50 毫升 Falcon 管中轻轻吹打。
$\mathrm{△}$$\mathrm{△}$/_\\triangle CRITICAL STEP Ensure that the cells are triturated gently and dissociated to single cells. Large clumps will reduce transfection efficiency.
关键步骤 确保细胞得到轻柔的研磨并解离成单细胞。大的细胞团块会降低转染效率。
21 Take a 10- $\mu \mathrm{l}$$\mu \mathrm{l}$mul\mu \mathrm{l} aliquot from the cell suspension and dilute it into $90\mu \mathrm{l}$$90\mu \mathrm{l}$90 mul90 \mu \mathrm{l} of D10 medium for counting. Count the cells and calculate the number of cells and the
从细胞悬浮液中取 10- $\mu \mathrm{l}$$\mu \mathrm{l}$mul\mu \mathrm{l} 的等分试样，并稀释到 $90\mu \mathrm{l}$$90\mu \mathrm{l}$90 mul90 \mu \mathrm{l} 的 D10 计数培养基中。计数细胞并计算细胞数量和
volume of suspension needed for transfection. We typically transfect $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} cells per condition with the Amaxa SF cell line 4D-Nucleofector X kit S, and we recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. Transfer the volume needed ( 20 ll per transfection plus waste volume) into a new Falcon tube.
转染所需的悬浮液量。我们通常对每个条件使用 Amaxa SF 细胞株 4D-Nucleofector X 试剂盒 S 转染 $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} 个细胞，我们建议计算比所需细胞数量多 20% 的细胞，以便在后续移液步骤中调整体积损失。将所需的体积（每次转染 20 ll 加废液量）转移到新的 Falcon 管中。
22 Spin down the cells from Step 21 at 200 g for 5 min at room temperature.
22 将步骤 21 中的细胞在 200 g 下于室温离心 5 分钟。
23 Prepare the transfection solution by mixing the SF solution and S1 supplement supplied in the Amaxa SF cell line 4D-Nucleofector X kit S; a total of $20\mu \mathrm{l}$$20\mu \mathrm{l}$20 mul20 \mu \mathrm{l} of supplemented SF solution is used per transfection. Likewise, we recommend calculating for $20\mathrm{\%}$$20\mathrm{\%}$20%20 \% more volume than required.
23 使用 Amaxa SF 细胞系 4D-Nucleofector X 试剂盒 S 中提供的 SF 溶液和 S1 补充剂混合制备转染溶液；每次转染使用总共 $20\mu \mathrm{l}$$20\mu \mathrm{l}$20 mul20 \mu \mathrm{l} 补充的 SF 溶液。同样，我们建议计算出比所需量多 $20\mathrm{\%}$$20\mathrm{\%}$20%20 \% 的体积。
24 Remove the medium completely from the pelleted cells from Step 22, and gently resuspend the cells in an appropriate volume ( $20\mu \mathrm{l}$$20\mu \mathrm{l}$20 mul20 \mu \mathrm{l} per $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} cells) of S1supplemented SF solution. Do not leave the cells in SF solution for extended periods of time.
24 从步骤 22 中的压片细胞中完全去除培养基，并用适量（每 $20\mu \mathrm{l}$$20\mu \mathrm{l}$20 mul20 \mu \mathrm{l} 个细胞 $2\times {10}^5$$2\times {10}^5$2xx10^(5)2 \times 10^{5} 毫升）S1 补充的 SF 溶液轻轻重悬细胞。 不要将细胞长时间置于 SF 溶液中.
$25$$25$25quad25 \quad Pipette $20\mu \mathrm{l}$$20\mu \mathrm{l}$20 mul20 \mu \mathrm{l} of resuspended cells into each DNA premix from Step 19. Pipette gently to mix and transfer to a Nucleocuvette strip chamber. Repeat this step for each transfection condition.
将复悬细胞的 $25$$25$25quad25 \quad 吸取至每个 Step 19 的 DNA 预混液中。轻轻吹打混匀后转移至 Nucleocuvette 槽室。对每个转染条件重复此步骤。
26 Electroporate the cells by using the Nucleofector 4D program recommended by Amaxa, CM-130.
使用 Amaxa 推荐的 Nucleofector 4D 程序 CM-130 对细胞进行电穿孔。
27 Gently and slowly pipette $100\mu \mathrm{l}$$100\mu \mathrm{l}$100 mul100 \mu \mathrm{l} of warm D10 medium into each Nucleocuvette strip chamber, and transfer all the volume into a well with the prewarmed medium from Step 18.
缓缓地和小心地移液 $100\mu \mathrm{l}$$100\mu \mathrm{l}$100 mul100 \mu \mathrm{l} 暖 D10 培养基到每个 Nucleocuvette 试剂条室，并将所有体积转移到上一步中预温培养基的孔中。

A CRITICAL STEP Cells are very fragile at this stage, and harsh pipetting can cause cell death.
在这一阶段，细胞非常脆弱，强力移液会导致細胞死亡。
28 Incubate the mixture for 24 h . At this point, transfection efficiency can be estimated from the fraction of fluorescent cells in the positive transfection control. Nucleofection typically results in $>70-80\mathrm{\%}$$>70-80\mathrm{\%}$> 70-80%>70-80 \% transfection efficiency.
### 28 混合物应孵育 24 小时。此时，可以从阳性转染对照中荧光细胞的比例来估算转染效率。Nucleofection 通常导致 $>70-80\mathrm{\%}$$>70-80\mathrm{\%}$> 70-80%>70-80 \% 转染效率。

29 Slowly add 1 ml of warm D10 medium to each well without dislodging the cells. Puromycin selection can be applied at a concentration of $1-3\mu {\mathrm{ml}}^{-1}$$1-3\mu {\mathrm{ml}}^{-1}$1-3muml^(-1)1-3 \mu \mathrm{ml}^{-1} for HEK 293FT cells (may vary depending on the cell line). Incubate the cells with puromycin for at least 72 h . Cells can then be cultured in regular medium for downstream experiments or harvested for genotyping.
将 1 ml 温热的 D10 培养基缓慢地逐个添加至各孔内，注意不要使其脱落。HEK 293FT 细胞的嘌呤霉素选择浓度可以是 $1-3\mu {\mathrm{ml}}^{-1}$$1-3\mu {\mathrm{ml}}^{-1}$1-3muml^(-1)1-3 \mu \mathrm{ml}^{-1} （根据不同的细胞系可能有所不同）。将细胞与嘌呤霉素一起培养至少 72 小时。之后可在常规培养基中培养用于下游实验或收集用于基因分型。

A CRITICAL hESCs and human induced pluripotent stem cells can vary widely in their transfection efficiency, tolerance of single-cell dissociation and maintenance conditions. For a given cell line of interest, relevant literature or the distributor should be consulted.
A CRITICAL hESCs 和人类诱导多能干细胞的转染效率、耐受单细胞解离和保持条件可能存在很大差异。 对于给定的感兴趣细胞系，应参考相关文献或供应商。
30 Maintaining HUES9 cells. We routinely maintain HUES9 cells (a hESC cell line) in feeder-free conditions with mTesR1 medium. Prepare mTeSR1 medium
保持 HUES9 细胞。我们采用无饲养层条件下，使用 mTesR1 培养基来常规维护 HUES9 细胞（一种 hESC 细胞系）。配制 mTeSR1 培养基