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【求助】急求INS-1细胞的培养，请养过的战友进！
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说出来不怕丢人，我养INS-1细胞已经买过四次细胞了，每次买回来还好好的，就是一传代就不行，所以左问又问，决定不信这个邪，决定再买一株，这次从公司买的细胞，刚寄过来的时候还是很好（快递过来没脱壁，细胞形态也还好），根据公司的建议，将0.25%胰酶-0.38%EDTA稀释五倍，消化下来后离心后加培养基培养。好，听他们的。继续用他们寄细胞的培养基，胰酶稀释五倍后消化，待细胞变圆还未脱壁时培养基终止，250g离心3分钟（大概1000转），加培养基分瓶。第二天看，两瓶细胞都有一些死细胞漂浮在培养基中，贴壁的细胞看起来不是很漂亮，第三天死细胞更多，状态更加不好，细胞大小不均一，还有一些贴壁不牢。根据以往的经验，细胞可能不会长了，最后逐渐脱壁死亡。有两个问题：1.公司建议的培养基中除了加10% fetal calf serum，100 U/ml penicillin, 100 ｕg/ml streptomycin, 10 mmol/l HEPES, 2mmol/l L-glutamine, 1 mmol/l sodium pyruvate, and 50 ｕmol/l b-mercaptoethanol以外，还另外加了5.6mmol/L的葡萄糖，奇怪，Gibco和Hyclone的1640都是含11.1mmol/L的葡萄糖，不应该另外加了，问公司，他们说他们说明书是从引进的细胞库拿过来的，而且他们一直是这样养的也没事，想来也是，16.7mmol/L的葡萄糖从复苏到快递到我手里至少也有好4，5天，高糖诱导的凋亡早就应该开始了，到我手里不可能有这么好的状态，而且我还测了一下培养基的葡萄糖：12.8mmol/L。问题：和糖的浓度有没有关系？2.传代到底有什么问题，稀释五倍的胰酶在时间上还是很好控制，而且还离了心，应该去除了EDTA 的影响，百思不得其解，为什么传代后就是不行。胰酶所用的原装丹麦进口Gibco胰酶。3.这个细胞有这么难伺候吗？我已经被折磨得不行了？战友帮我分析分析，有什么经验介绍给我！
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不知道你最近有没有养起来，我也经常遇到这样的问题，一般还是因为消化的问题
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关于丁香园The Preparation of Sodium Pyruvate on JSTOR
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The Preparation of S...
Journal Article
William v.B. Robertson
New Series, Vol. 96, No. 2482 (Jul. 24, 1942), pp. 93-94
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Stable URL: http://www.jstor.org/stable/1670103
Page Count: 2
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Processing your request...关注今日：70 | 主题：526045
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【求助】DMEM中加有sodium pyruvate的原因
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这个帖子发布于9年零48天前，其中的信息可能已发生改变或有所发展。
在培养基中为什么要加入sodium pyruvate，请高人们指点
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加丙酮酸钠是为满足一些细胞的代谢需要，比如做杂交瘤实验。丙酮酸钠可以作为细胞培养中的替代碳源，尽管细胞更倾向于以葡萄糖作为碳源，但是，如果没有葡萄糖的话，细胞也可以代谢丙酮酸钠。
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CRISPR/Cas9对CAR-T细胞进行多重基因编辑
发布时间：日
用CRISPR/Cas9对CAR-T细胞进行多重基因编辑
Xiaojuan Liu1,&*, Yongping Zhang2,&*, Chen Cheng1, 3,&*,&Albert W Cheng4, Xingying Zhang1, 5, Na Li1, Changqing Xia2, 6, Xiaofei Wei7, Xiang Liu1, Haoyi Wang1, 5, 8&&
1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of&Zoology, The Chinese Academy of Sciences, Beijing, C&
2Department&of Hematology, Xuanwu Hospital, Capital Medical University, Beijing,&C&
3Graduate School, University of Science and Technology of China,&Hefei, C
4The Jackson Laboratory for Genome Medicine, Farmington,&CN, USA;&
5University of Chinese Academy of S&
6Department of&Pathology, Immunology and Laboratory Medicine, University of Florida,&FL, USA;&
7Beijing Cord Blood Bank, Beijing, C&
8The Jackson&Laboratory, Bar Harbor, ME, USA
实验材料：
Fresh umbilical cord blood (UCB) units
分离脐带血T细胞
Beijing Cord Blood Bank (Beijing, China)
Histopaque-1077
密度梯度离心
&(Sigma-Aldrich)
EasySep human T cell enrichment kit
&(Stemcell Technologies)
anti-CD3/anti-CD28 Dynabeads
&(ThermoFisher Scientific)
X-vivo15 medium&
heat-inactivated fetal bovine serum
血清，培养T细胞和各种细胞系
(ThermoFisher Scientific)
L-glutamine
谷氨酰胺，培养T细胞和各种细胞系
(ThermoFisher Scientific)
sodium pyruvate
丙酮酸钠，培养T细胞和各种细胞系
(ThermoFisher Scientific)
recombinant human IL-2
白介素2，激活T细胞
(ThermoFisher Scientific)
Trypan blue
台盼蓝，细胞计数
&(ThermoFisher Scientific)
淋巴瘤细胞系
(Burkitt&s lymphoma cell line, ATCC-CCL86)
B细胞淋巴瘤细胞系
(B lymphoblast cell line, ATCC-CCL213)
K562 cells&
人红白血病细胞系
(human erythroleukemic cell line, ATCC-CCL243)
48-well plates
&(Corning)
firefly luciferase lentiviral particles
荧光素酶慢病毒
Genechem (Shanghai, China)
K562细胞分选
Beckman Coulter Inc
RPMI1640 medium&
Raji/Daudi/K562细胞系培养
(ThermoFisher Scientific)
(ATCC-CRL3216)
培养293T细胞，加10%血清
(ThermoFisher Scientific)
pMD2.G, psPAX2&
包装病毒的质粒
Lipofectamine3000&
转染293T包装病毒
(ThermoFish Scientific)
ultracentrifugation&
超速离心分离病毒
(Merck Millipore)
pX330 plasmid&
sgRNA编码质粒
(Addgene plasmid #4223)
MEGAshortscript T7 kit&
体外转录得到sgRNA
(ThermoFisher Scientific)
MEGAclear columns&
(ThermoFisher Scientific)&
Cas9 protein&
(ThermoFisher Scientific)
4D-Nucleofector System N&
P3 Primary Cell 4D Nucleofector X Kit
核转试剂盒，转T细胞
(Lonza)&V4XP-3024&
ThermoFisher Scientific
(Beckman Coulter Inc)
TCR &/&-PE/Cy7&
(IP26, Biolegend),&
TCR&/&-PE&
(IP26, Biolegend),&
&2-microglobulin (B2M)-PE &
(2M2, Biolegend),
&2-microglobulin (B2M)-FITC&
(2M2, Biolegend),&
CD279(PD-1)-APC&
(EH12.2H7, Biolegend),&
CD279(PD-1)-PE&
(EH12.2H7, Biolegend),&
HLA-A2-FITC&
(BB7.2, BD Pharmingen)
surveyor mutation detection kit&
突变检测试剂盒
(Integrated DNA Technologies, Inc)
pEASY Blunt Cloning Kit&
平末端克隆
(Transgen Biotech)
EasySep PE selection kit&
分离CAR-T细胞
(Stemcell Technologies)
检测白介素和干扰素&
(Biolegend)
Celltrace Violet&
细胞坏死检测
(ThermoFisher Scientific)
FITC-AnnexinV and 7-AAD&
染色后做流式
(Biolegend)&
6-12-week-old NOD-Prkdcscid Il2rgnull (NPG) mice&
(VITALSTAR, Beijing, China)
matrigel matrix&
混合肿瘤细胞，做活体注射
Xenogen IVIS Imaging System&
活体成像系统
(Perkin Elmer Life Sciences)
d-luciferin&
活体成像底物
(Perkin Elmer Life Sciences)
实验方法：
从脐带血中分离并扩增T细胞 Isolation and expansion of T cells from UCB units
Fresh umbilical cord blood (UCB) units&were obtained from healthy volunteer donors who had provided informed consent from the&Beijing Cord Blood Bank (Beijing, China), and mononuclear cells were separated using density gradient centrifugation with&Histopaque-1077 (Sigma-Aldrich).&T cells were isolated using the&EasySep human T cell enrichment kit (Stemcell Technologies), activated and expanded with&anti-CD3/anti-CD28 Dynabeads (ThermoFisher Scientific)&at a bead to T cell ratio of 1:1 according to the manufacturer's instructions. UCB-derived T cells were cultured in&X-vivo15 medium (Lonza)&supplemented with 5% (v/v)&heat-inactivated fetal bovine serum, 2mM&L-glutamine, and 1mM sodium pyruvate in the presence of 300 IU/mL recombinant human IL-2 (all from ThermoFisher Scientific). Viable cells were enumerated using&Trypan blue (ThermoFisher Scientific)&exclusion. All cells were grown at 37 &C in a 5% CO2 atmosphere. &
细胞系 Cell lines
The following CD19-expressing immortalized cell lines were used:&Raji (Burkitt&s lymphoma cell line, ATCC-CCL86),Daudi (B lymphoblast cell line, ATCC-CCL213), and&K562-CD19. Rajiffluc cells for bioluminescent imaging were generated by transfection of Raji cells with an expression cassette for&firefly luciferase lentiviral particles from Genechem (Shanghai, China). To isolate a stable line expressing firefly luciferase (Raji-ffluc), transfected cells underwent puromycine selection and single cell cloning. K562-CD19 cells expressing CD19 were prepared by lentiviral transfection of&K562 cells (human erythroleukemic cell line, ATCC-CCL243)&with an expression cassette for CD19. The cDNA for full-length CD19 derived from Daudi cells was cloned into the FUW lentiviral backbone. Lentivirus-containing supernatant was prepared and K562 cells were transduced with this supernatant and then CD19-expressing cells were sorted by&flow cytometry (MoFlo XDP, Beckman Coulter Inc)&and underwent single cell cloning to obtain a population of K562 cells that uniformly expressed high levels of CD19. All above cell lines&were maintained in&RPMI1640 medium (ThermoFisher Scientific). Lentiviral producer cell lines&293T (ATCC-CRL3216)were maintained in&DMEM (ThermoFisher Scientific). All media were supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin and streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. All cell lines were grown at 37 &C in a 5% CO2 atmosphere. &
制备 CAR-T细胞 CAR-T generation&
Second-generation CD19 CARs were constructed using single-chain variable fragment (scFv) derived from antibody clones FMC63, hinge and transmembrane regions from CD8&, intracellular domain from CD137, and intracellular domain from CD247 (CD3-zeta) [1]. The CAR sequence is followed in frame by the 2A ribosomal skipping sequence and eGFP sequence. The final CD19CAR-2A-eGFP was cloned into the FUW lentiviral vector backbone downstream from an EF1& promoter (Fuw-EF1&-CD19CAR-2A-eGFP). Lentiviruses were produced by cotransfecting 293T cells with Fuw-EF1&-CD19CAR-2A-eGFP and packaging plasmids&pMD2.G, psPAX2 (addgen)using&Lipofectamine3000 (ThermoFish Scientific). Virus supernatants were harvested on days 2 and 3 and concentrated by&ultracentrifugation (Merck Millipore). UCBderived T cells were transduced with the supernatant and CAR+ T cells were identified by eGFP expression. &
体外转录制备sgRNA Generating sgRNAs using in vitro transcription&
We used oligonucleotides containing T7 promoter and 20bp targeting sequences as forward primer, and an sgRNA backbone reverse primer to amplify sgRNA-coding fragment using&pX330 plasmid (Addgene plasmid #4223)&as template. The T7-sgRNA PCR products were gel-purified and used as the template for IVT using&MEGAshortscript T7 kit (ThermoFisher Scientific). RNAs were purified with&MEGAclear columns (ThermoFisher Scientific)&and eluted in RNase-free water. &
制备TCR/B2M双敲除和TCR/B2M/PD-1三敲除的CAR-T细胞&
Generation of TCR/B2M double knockout (DKO) and TCR/B2M/PD-1 triple knockout (TKO) CAR-T cells &
Freshly purified primary T cells were activated for 3 days according to the procedure described above, and then transduced with lentiviral vectors harboring the CD19 CARs. Two days after transduction, CAR-T cells were electroporated with&Cas9 protein (ThermoFisher Scientific)&and the intended sgRNAs targeting the TCR& constant chain (TRAC), &2-microglobulin (B2M) exon1, andPD-1 exon1 by&4D-Nucleofector System N (Lonza) using the&P3 Primary Cell 4D&Nucleofector X Kit, V4XP-3024 (Lonza). Cas9:single-guide RNA ribonucleoproteins (Cas9RNPs) were prepared immediately before experiments by incubating Cas9 protein with sgRNA at a 1:1 ratio at room temperature for 10 min. 3&106 cells were washed twice with&Dulbecco&s Phosphate Buffered Saline (DPBS, ThermoFisher Scientific)&by centrifuging at 200g for 5 minutes and resuspended in 100 &L transfection buffer containing Cas9 RNP and then transferred into the electroporation cuvette. Program EO-115 was selected for high efficiency. After electroporation, cells were resuspended in 2 mL pre-warmed T cell medium and transferred into a 12-well cell plate and incubated at 37 &C in 5% CO2. The transfection efficiency was evaluated 3 and 7 days after electroporation. Cell culture medium was half replaced by fresh complete medium every 2 ~ 3 days. &
流式细胞术 Flow cytometry&
CytoFLEX (Beckman Coulter Inc)&was used to perform fluorescent expression analysis. Cells were harvested on the following days after transfection and stained with mouse anti-human antibody labeled by fluorescence for 10 minutes at room temperature in the dark as follows:&TCR &/&-PE/Cy7 (IP26, Biolegend),&TCR&/&-PE (IP26, Biolegend),&&2-microglobulin (B2M)-PE (2M2, Biolegend),&&2-microglobulin (B2M)-FITC (2M2, Biolegend),&CD279(PD-1)-APC (EH12.2H7, Biolegend),&CD279(PD-1)-PE (EH12.2H7, Biolegend),&HLA-A2-FITC (BB7.2, BD Pharmingen). &
Surveyor nuclease assay and sequencing&
The levels of genomic disruption of TRAC, B2M, PD-1 in T cells or CAR-T cells were determined by surveyor nuclease assay using&surveyor mutation detection kit (Integrated DNA Technologies, Inc). The percentage target disruption was quantified by densitometry and calculated as described [2]. The PCR products were also sequenced for TIDE (Tracking of Indels by Decomposition) analyses using specially designed software provided as a simple web tool (available at http://tide.nki.nl). The PCR primers used for the amplification of target loci and sequencing are listed in Supplementary Table 1. The purified PCR products were ligated with pEASY blunt cloning vector usingpEASY Blunt Cloning Kit (Transgen Biotech)&to detect mutant alleles. Ligation products were used for transformation and about 20-30 colonies per sample are sequenced using universal primer M13F. &
富集双敲除好三敲除的CAR-T细胞 Enrichment of DKO and TKO CAR-T cells&
DKO and TKO CAR-T Cells were enriched using&EasySep PE selection kit (Stemcell Technologies)&according to the manufacturer's instructions. Briefly, the gene modified CAR-T cells were labeled with PE-conjugated antibody (TCR&/&-PE, &2-microglobulin-PE, PD-1-PE) and anti-PE MicroBeads, and then the labeled cells were put into a magnetic field. Using this procedure, the magnetically labeled PE-positive cells were retained in the tube while the unlabeled DKO and TKO CAR-T cells could be recovered in the supernatant. &
ELISA Cytokine enzyme-linked immunosorbent assay (ELISA) &
Cytokine production by effector (CAR-T, DKO CAR-T, TKO CAR-T, T) cells was evaluated by co-incubation with target tumor cells (Daudi, Raji, K562-CD19, K562) at a 1:1 ratio (104 cells each) for 24 hours. Supernatants were harvested and&IL-2 and IFN-& levels were analyzed by ELISA (Biolegend). &
流式细胞毒检测 Flow-based cytotoxicity assay&
The cytolytic activity and specificity of CAR-T cells were assessed according to the flow cytometry-based cytotoxicity assay described in [3]. Lytic activities of effector cells were tested by Violet/AnnexinV and 7-AAD labeling cytotoxicity assay. Target tumor cells were labeled with 1 &M&Celltrace Violet (ThermoFisher Scientific)&for 25 min at 37&C in PBS. Labeling was stopped by adding 10 mL complete culture medium and incubated at 37 &C for 5 minutes and extensively washed in complete culture medium before seeding into the&48-well plates (Corning).&Violet-labeled target cells were then incubated with effector cells by different effector to target ratio for 4 hours.&FITC-AnnexinV and 7-AAD (Biolegend)&were added to determine the ratio of dead target cells. Samples were analyzed by flow cytometry. Target cells were selected by gating on the Violet-positive cell population and further analyzed for different subpopulations. The percentages of cytotoxic activity was calculated using the following equation: %specific cell death={[%(Violet+AnnexinV++Violet+ AnnexinV-7-AAD+)-%spontaneous (Violet+AnnexinV++Violet+ AnnexinV-7-AAD+)]/[100%-% spontaneous (Violet+Annexin V++Violet+ AnnexinV-7-AAD+)]}&100% &
在体实验 In vivo studies &
6-12-week-old NOD-Prkdcscid Il2rgnull (NPG) mice (VITALSTAR, Beijing, China)&were injected with 2&105 Raji-ffluc cells via intraperitoneal injection in a volume of 50&L DPBS and 50&L&matrigel matrix (Corning). Two days after injection, tumor engraftment was evaluated by serial biophotonicimaging using the&Xenogen IVIS Imaging System (Perkin Elmer Life Sciences). Mice&were injected intraperitoneally with 3 mg&d-luciferin (Perkin Elmer Life Sciences), and then imaged 4 minutes later with an exposure time of 30 seconds. Luminescence images were analyzed usingLiving Image software (Perkin Elmer Life Sciences). The bioluminescence signal was measured as total photon flux normalized for exposure time and surface area and expressed in units of photons/s/cm2/steradian (p/s/cm2/sr). Mice with progressively growing tumors were segregated into treatment groups bearing comparable tumor loads and received 200 uL DPBS /mouse, 5x106 T cells /mouse, 5x106CAR-T cells /mouse, 5x106 DKO CAR-T cells /mouse intraperitoneally one day later. The tumor loads were evaluated 7 days after treatment. &
脱靶分析 Off target analysis&
The potential off target of each sgRNA is predicted using Benchling software using algorithm described in [4]. The top five targets for each sgRNA were amplified by PCR and subjected to Sanger sequencing. Sequencing results were analyzed using the TIDE method [5].&
In exome sequencing experiments, we used NimbleGen SeqCap_EZ_Exome_v3+UTR exonic target sequences to capture and enrich human exonic region following the manufacture&s instruction. Briefly, we first built the DNA library and randomly fragmented DNA. The DNA fragments were then hybridized with the exome lipid chip forming complex. After hybridization, the library was purified, evaluated for its quality, and applied to sequencing. We used genomic alignment software (BWA [6]) to map the clean reads to the reference genome UCSChg19 and samtools [7] to sort the BAM file for mutation detection with high accuracy. Potential sequence variations were called using mutational analysis software GATK [8] against the hg19 genome and were then filtered by quality value, depth and reproducibility using default parameters. Indels called in the different samples were overlapped and presented as Venn diagrams. 100 bp window of genomic sequences surrounding called indels were extracted. All 20 mer sequences followed by NGG PAM from both strands were enumerated and aligned to the sgRNA spacers. The best alignment (with minimal number of mismatches) of each sgRNA spacer against each 100 bp window was reported with the number of mismatches. &
统计分析 Statistical analysis&
Graphpad Prism 5.0 (Graphpad software, San Diego, CA) was used for all statistical analysis. The mean & S.E.M. was determined for each treatment group in the individual experiments. The onetailed Student t-test was used to determine the significances between treatment and control groups. P-values & 0.05 were significant.&
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