ImmunoCult™ XF培养基

cGMP，无血清和无异源培养基，用于人免疫细胞扩增

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产品号 #100-0956_C

cGMP，无血清和无异源培养基，用于人免疫细胞扩增

产品优势

使用符合相关cGMP标准的培养基稳定扩增免疫细胞，用于临床应用
在无血清和无异源培养条件下扩增细胞，减少实验间差异
实现具有高活力的稳定T细胞扩增
获得在重新刺激后能够产生细胞因子的T细胞
与ImmunoCult™人T细胞激活剂搭配使用，实现无磁珠激活T细胞

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总览

使用高性能、符合cGMP标准的ImmunoCult™-XF培养基，支持人T细胞的培养并助力您的细胞疗法开发。该培养基专为临床应用中的高效扩增而设计，无需血清或血清衍生物，不含酚红、无异源成分且未添加细胞因子——为您的工作流程提供完全的灵活性。T细胞在培养仅10-12天后即可收获，扩增后的T细胞能够产生包括干扰素γ (IFN-γ)和白细胞介素4 (IL-4) 等的细胞因子。该即用型培养基根据现行GMP规范生产，确保高质量即高度一致性。

ImmunoCult™-XF可以单独使用，或与ImmunoCult™人CD3/CD28 T细胞激活剂或ImmunoCult™ 人CD3/CD28/CD2 T细胞激活剂结合使用，以温和、无磁珠的方式激活T细胞。有关使用ImmunoCult™扩增T细胞的更多信息，请查阅产品说明书。

如需申请ImmunoCult™-XF的FDA主文件授权书（LOA），请点击此处。

实验数据

Culture Protocol for T Cell Activation And Expansion with ImmunoCult™-XF

Figure 1. Culture Protocol for T Cell Activation and Expansion

(A) T cells were isolated from fresh leukopaks (Catalog #70500) from healthy donors using EasySep™ Human T Cell Isolation Kit (Catalog #17951) and cryopreserved in CryoStor® CS10 (Catalog #07930). On Day 0, T cells were thawed, washed, and resuspended at 1 x 10⁶ cells/mL in ImmunoCult™-XF (Catalog #100-0956) supplemented with 180 IU/mL Human Recombinant IL-2, ACF (rhIL-2; Catalog #78145). Cells were seeded into 24-well tissue culture plates (1 mL/well) and stimulated with 25 µL/mL ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785)*. On Days 3, 5, 7, and 10, cells were re-seeded in fresh ImmunoCult™-XF + rhIL-2 at low or high cell densities as outlined in the table (B). Cells were harvested on Day 12.

*A similar protocol was performed using ImmunoCult™ Human CD3/CD28 T Cell Activator (Catalog #100-0784). Please reach out to our Product and Scientific Support team for additional information.

Figure 2. ImmunoCult™ Reagents Result in Robust T Cell Expansion Under Low Cell Density Culture Conditions

T cells were cultured with ImmunoCult™-XF (Catalog #100-0956), Human Recombinant IL-2, ACF (Catalog #78145), and ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785) for 12 days following the low cell density protocol described in Figure 1. Cells were harvested and analyzed at different time points. Cell count and viability were assessed using the NucleoCounter® NC-250™ automated cell analyzer, and cell surface marker expression was analyzed by flow cytometry. (A) Points and bars in the graph represent T cell viability and average cumulative fold expansion, respectively, for each assessed culture time point. An average viability of 92.8 ± 1.8% and a total fold expansion of 1497 ± 143 were achieved by Day 12 in the low cell density cultures. Data represent mean ± SEM (n = 11). (B) The ratio of CD4+ T cells to CD8+ T cells (CD4:CD8) was comparable between Day 0 and Day 12 in 10 out of 11 donors. (C) The frequency of T cells expressing PD-1, TIM-3, and LAG-3 was upregulated upon stimulation and subsequently decreased to basal levels (n = 4). (D) The frequency and number of T cell subtypes in CD4+ and CD8+ populations during the 12-day culture period are shown. Cell numbers were calculated based on 10⁶ starting T cells. The number of central memory T cells remained constant from day 7 to 12. T cell subsets: naive and T memory stem cell (T_N/SCM) CCR7+CD45RO-; central memory (T_CM) CCR7+CD45RO+; effector memory (T_EM) CCR7-CD45RO+; effector cells (T_EFF) CCR7-CD45RO- (n = 4 - 11).

Figure 3. ImmunoCult™ Reagents Support T Cell Activation and Expansion Following High Cell Density Protocol

T cells were cultured with ImmunoCult™-XF (Catalog #100-0956), Human Recombinant IL-2, ACF (Catalog #78145), and ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785) for 12 days following the high cell density protocol described in Figure 1. Cells were harvested at different time points and analyzed. Cell count and viability were assessed using the NucleoCounter® NC-250™ automated cell analyzer, and cell surface marker expression was analyzed by flow cytometry. (A) Points and bars in the graph represent T cell viability and average cumulative fold expansion, respectively, for each assessed culture time point. An average viability of 89.3 ± 2.3% and a total fold expansion of 67 ± 7 were achieved by Day 12 in the high cell density cultures. Data represent mean ± SEM (n = 4). (B) The ratio of CD4+ T cells to CD8+ T cells (CD4:CD8) was measured at the beginning and end of culture. The CD4:CD8 ratios increased in two out of four donors tested. (C) The frequency of T cells expressing PD-1, TIM-3, and LAG-3 increased upon stimulation and subsequently decreased to basal levels by Day 12 of culture (n = 4), with the exception of LAG-3 expression in the CD8⁺ population. (D) Data shows the frequency and number of T cell subtypes in CD4+ and CD8+ populations at the indicated time points. Cell numbers were calculated based on 10⁶ starting T cells. The number of central memory T cells continued to increase from Day 7 to Day 12 of culture. T cell subsets: naive and T memory stem cells (TN/SCM) CCR7+CD45RO-; central memory (TCM) CCR7+CD45RO+; effector memory (TEM) CCR7-CD45RO+; effector cells (TEFF) CCR7-CD45RO- (n = 4).

Culture Protocol for Expansion of T Cells with ImmunoCult™ Reagents in the Xuri™ Cell Expansion System W25

Figure 4. Culture Protocol for Expansion of T Cells with ImmunoCult™ Reagents in the Xuri™ Cell Expansion System W2

T cell expansion workflow in Xuri™ Cell Expansion System W25: T cells were isolated from fresh leukopaks (Catalog #70500) using the EasySep™ Human T Cell Isolation Kit (Catalog #17951) and cryopreserved in Cryostor® CS10 (Catalog #07930). On Day 0, cryopreserved cells were thawed and resuspended at 1 x 10⁶ cells/mL in ImmunoCult™-XF (Catalog #100-0956) supplemented with 180 IU/mL Human Recombinant IL-2, ACF (Catalog #78145). 5 x 10⁷ T cells were seeded in a T175 tissue culture flask and activated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785) at 25 μL/mL. On Day 3, cell density was adjusted to 1 - 2.5 x 10⁵ cells/mL by adding fresh ImmunoCult™-XF with IL-2. On Day 5, the cell suspension containing 3 x 10⁸ cells/L of culture volume was inoculated into a Xuri™ 2L Cellbag™ Bioreactor and cultured until Day 10. Once the cell density reached 1.5 x 10⁶ cells/mL, perfusion was initiated at 0.5 L/day on Day 7 and increased to 1 L/day on Day 9. The culture was harvested on Day 10.

Figure 5. Expansion of T Cells with ImmunoCult™ Reagents in the Xuri™ Cell Expansion System W25

T cells were cultured with ImmunoCult™-XF (Catalog #100-0956), Human Recombinant IL-2, ACF (Catalog #78145), and ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785) for 10 days following the protocol described in Figure 4. (A) Points and bars in the graph represent T cell viability and average cumulative fold expansion, respectively, for each assessed culture time point. On average, 92.7 ± 0.7% viability and a total fold expansion of 503 ± 51 were achieved by Day 10. Data represent mean ± SEM (n = 4). (B) Changes in the ratio of CD4+ T cells to CD8+ T cells (CD4:CD8) were measured at the beginning and end of culture. Some variability among donors was observed (n = 4). (C) The frequency of T cells expressing PD-1, TIM-3, and LAG-3 increased by Day 3 and subsequently decreased from Day 3 to Day 10 (n = 4). (D) The frequency and number of T cell subtypes in CD4+ and CD8+ populations are shown. Cell numbers were calculated based on 10⁶ starting T cells. The number of central memory T cells peaked on Day 7 of the culture. T cell subsets: naive and T memory stem cells (TN/SCM) CCR7+CD45RO-; central memory (TCM) CCR7+CD45RO+; effector memory (TEM) CCR7-CD45RO+; effector cells (TEFF) CCR7-CD45RO- (n = 4). (E) The frequencies of expanded T cells expressing IFN-ɣ and TNF-ɑ upon stimulation with PMA and ionomycin were 66.6 ± 2.1% and 96.3 ± 1.1%, respectively, in the CD4+ population, and 73.2 ± 11.4% and 93.8 ± 1.3%, in the CD8⁺ population. Additionally, the frequencies of expanded CD8+ T cells expressing perforin and granzyme B without stimulation were 23.3 ± 9.1% and 37.7 ± 6.8%, respectively (n = 4).

Figure 6. High-Efficiency TRAC Knockout of T Cells

(A) The protocol for non-viral genetic modification and expansion of T cells: On Day 0, T cells were isolated from fresh leukopaks using the EasySep™ Human T Cell Isolation Kit (Catalog #17951). Cells were resuspended at 1 x 10⁶ cells/mL in ImmunoCult™-XF (Catalog #100-0956) supplemented with 180 IU/mL Human Recombinant IL-2, ACF (rhIL-2; Catalog #78145). Cells were seeded into 24-well tissue culture plates at 1 mL/well and stimulated with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785) at 25 μL/mL. Following 3 days of activation, RNP complexes containing ArciTect™ Cas9 Nuclease (Catalog #76004) and sgRNA targeting the TRAC gene were delivered to T cells by electroporation (Neon® Transfection System). Cells were then returned to culture in ImmunoCult™-XF supplemented with rhIL-2 for another 9 days of expansion following the low cell density protocol. (B) Representative flow cytometry plots of TCRαβ and CD3 expression analysis on Days 2 and 9 post-T cell editing. (C) The gene editing efficiency was assessed by measuring the frequency of the remaining TCRαβ+CD3+ cells in the mock (91.8 ± 1.1%) and edited T cells (16.1 ± 2.6%) 2 days post-editing. The frequency of TRAC-edited cells was maintained throughout the culture duration. (D) The viability of T cells was measured using the NucleoCounter® NC-250™ automated cell analyzer across culture periods. On Day 3, the viability of T cells further decreased immediately following transfection. (E) Cumulative cell expansion post-electroporation was comparable between mock-treated and TRAC-edited T cells, suggesting that the edited T cells are functional. Data represent mean ± SEM (n = 3).

Figure 7. ImmunoCult™ NK Cell Base Medium and GMP ImmunoCult™-XF Support Natural Killer Cell Expansion

Natural killer (NK) cells were isolated from human peripheral blood samples using EasySep™ Human NK Cell Isolation Kit (Catalog #17955), then cultured in media supplemented with ImmunoCult™ NK Cell Expansion Supplement (Catalog #110-0715) for a total of 14 days. Cells were stimulated with ImmunoCult™ NK Cell Expansion Coating Material (Catalog #100-0714) on Days 0, 7, and 10 of culture. The medium used was either ImmunoCult™ NK Cell Base Medium (Catalog #100-0712) or GMP ImmunoCult™-XF (Catalog #100-0956). (A) NK cells undergo robust expansion over 14 days of expansion with ImmunoCult™ NK Cell Base Medium or GMP ImmunoCult™-XF. (B) NK cells remain highly viable after 14 days of expansion in ImmunoCult™ NK Cell Base Medium or GMP ImmunoCult™-XF. Each column with error bars represents the mean ± S.E.M. across three individual donors tested across three different manufactured lots of media.

产品说明书及文档

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ImmunoCult™-XF

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100-0956

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文献 (39)

High-Specificity CRISPR-Mediated Genome Engineering in Anti-BCMA Allogeneic CAR T Cells Suppresses Allograft Rejection in Preclinical Models Cancer Immunology Research 2024 Feb

Abstract

Allogeneic CAR T–cell therapies are being developed for hematologic malignancies. The authors implement a Cas12a chRDNA platform to generate allogeneic immune-cloaked BCMA-specific CAR T cells with resistance to host response–mediated rejection for evaluation in multiple myeloma. AbstractAllogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here,we describe preclinical data supporting the ongoing first-in-human clinical study,the CaMMouflage trial (NCT05722418),evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic,allogeneic anti–B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor–derived T cells using a Cas12a CRISPR hybrid RNA–DNA (chRDNA) genome-editing technology platform. To address allograft rejection,CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition,T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition,CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy,when combined with an immune-cloaking armoring strategy to dampen allograft rejection,offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst,p. 385

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The immunopathological landscape of human pre-TCRα deficiency: from rare to common variants M. Materna et al. Science (New York,N.Y.) 2024 Mar

Abstract

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-TCRα expression. Low circulating naïve αβ T cell counts at birth persisted over time,with normal memory αβ and high γδ T cell counts. Their TCRα repertoire was biased,suggesting that noncanonical thymic differentiation pathways can rescue αβ T cell development. Only a minority of these individuals were sick,with infection,lymphoproliferation,and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naïve αβ T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αβ T cells,autoimmune conditions were more frequent in these patients than in the general population.

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Delivery of a BET protein degrader via a CEACAM6-targeted antibody–drug conjugate inhibits tumour growth in pancreatic cancer models Y. Nakazawa et al. Nature Communications 2024 Mar

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy,we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC),a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody,#84.7,with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies,a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts,with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC. Subject terms: Pancreatic cancer,Drug development,Targeted therapies

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ImmunoCult™ XF培养基

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