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Cryostor cs10 cryopreservation medium

Manufactured by STEMCELL
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CryoStor CS10 is a cryopreservation medium designed for the freezing and storage of cells. It is formulated to provide optimal cryoprotection and cell viability upon thawing.

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Lab products found in correlation

Rosettesep human t cell enrichment kit, by STEMCELL (5 mentions) Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions) Retronectin, by Takara Bio (1 mentions) Anti pe microbeads, by Miltenyi Biotec (1 mentions) Mr frosty freezing container, by Thermo Fisher Scientific (1 mentions) Ficoll, by Merck Group (1 mentions) Leukopak, by STEMCELL (1 mentions) Imdm 10 ab human serum, by Thermo Fisher Scientific (1 mentions) Percoll, by GE Healthcare (1 mentions) Flow cytometric analysis, by BD (1 mentions) Whole blood column kit, by Miltenyi Biotec (1 mentions) Cellxvivo human b cell expansion kit, by R&D Systems (1 mentions)

8 protocols using cryostor cs10 cryopreservation medium

1

Isolation and Cryopreservation of Primary Human T Cells

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Healthy donor buffy coats were collected by and purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol using Lymphoprep density gradient medium and SepMate-50 tubes. Isolated T cells were cryopreserved at 2×107 T cells per vial in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).
Lynn R.C., Weber E.W., Sotillo E., Gennert D., Xu P., Good Z., Anbunathan H., Lattin J., Jones R., Tieu V., Nagaraja S., Granja J., DeBourcy C., Majzner R., Satpathy A.T., Quake S.R., Monje M., Chang H, & Mackall C.L. (2019). c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature, 576(7786), 293-300.
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2

Isolation and Activation of Primary Human T Cells

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Buffy coats were purchased from Stanford Blood Center under an IRB-exempt protocol, and processed using Lymphoprep density gradient medium and SepMate-50 tubes following manufacturer's instructions. Primary human T cells were positively selected using the RosetteSep Human T cell Enrichment kit (STEMCELL Technologies), and cryopreserved at 1–2 × 107 cells/vial in CryoStor CS10 cryopreservation medium (STEMCELL Technologies). Cryopreserved cells were thawed and activated the same day with CD3/CD28 Dynabeads (Gibco) at a 3:1 bead:cell ratio in T-cell media (RPMI1640 supplemented with 10% FBS, 10 mmol/L HEPES, 2 mmol/L GlutaMAX, 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 IU/mL IL2). Activated T cells were retrovirally transduced with CD93 CAR or cotransduced with CD93 CAR and CD19 iCAR on days 3 and 4 on Retronectin (Takara)-coated plates, and anti-CD3/CD28 beads were removed on day 5. Media and IL2 were changed every 2 to 3 days until day 10 or 11, when T cells were used for assays.
Richards R.M., Zhao F., Freitas K.A., Parker K.R., Xu P., Fan A., Sotillo E., Daugaard M., Oo H.Z., Liu J., Hong W.J., Sorensen P.H., Chang H.Y., Satpathy A.T., Majzner R.G., Majeti R, & Mackall C.L. (2021). NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity. Blood Cancer Discovery, 2(6), 648-665.
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3

Isolation and Cryopreservation of Primary Human T Cells

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Healthy donor buffy coats were collected by and purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol using Lymphoprep density gradient medium and SepMate-50 tubes. Isolated T cells were cryopreserved at 2×107 T cells per vial in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).
Lynn R.C., Weber E.W., Sotillo E., Gennert D., Xu P., Good Z., Anbunathan H., Lattin J., Jones R., Tieu V., Nagaraja S., Granja J., DeBourcy C., Majzner R., Satpathy A.T., Quake S.R., Monje M., Chang H, & Mackall C.L. (2019). c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature, 576(7786), 293-300.
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4

Isolation and Cryopreservation of CD39-Negative T Cells

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Healthy donor buffy coats were purchased from the Stanford Blood Center under an IRB-exempt protocol. Primary human T cells were isolated using the RosetteSep Human T cell Enrichment kit (Stem Cell Technologies) according to the manufacturer’s protocol. Isolated T cells were cryopreserved in CryoStor CS10 cryopreservation medium (Stem Cell Technologies).
CD39− T cells were purified using anti-PE MicroBeads (Miltenyi Biotec) and LD autoMACS (Miltenyi Biotec) columns according to the manufacturer’s protocol. Depletion efficiency was assessed by flow cytometry.
Klysz D.D., Fowler C., Malipatlolla M., Stuani L., Freitas K.A., Meier S., Daniel B., Sandor K., Xu P., Huang J., Labanieh L., Leruste A., Bashti M., Keerthi V., Mata-Alcazar J., Gkitsas N., Guerrero J.A., Fisher C., Patel S., Asano K., Patel S., Davis K.L., Satpathy A.T., Feldman S.A., Sotillo E, & Mackall C.L. (2023). Inosine Induces Stemness Features in CAR T cells and Enhances Potency. bioRxiv.
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5

Cryopreservation and Resuscitation of Epithelial Cells

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To cryopreserve the isolated cells, non-adherent cells containing epithelial cells and some mesenchymal cells were pooled from wells after the adherence step and pelleted at 200 × g for 4 min at RT. Media was removed and cells were resuspended in Cryostor CS10 cryopreservation medium (7930, Stem Cell Technologies, SCT, Cambridge, UK) containing 10 mM Y27632 (72304, SCT), at 1 × 106 cells/mL. Cryovials were stored overnight in a Mr Frosty freezing container (5100-0001, TFS) at −80 °C, and then transferred to liquid nitrogen for long-term storage.
To resuscitate the cells, the cryovials were thawed briefly in a water bath at 37 °C. The cell suspension was transferred to 5× volume of DMEM/F12 and pelleted by centrifugation at 200 × g for 4 min at RT and cultured as previously described. Cells were seeded at a concentration of 1–2 × 105 cells/well in 24-well transwells and CHIR99021 (S1263-SEL, Stratech Scientific, Ely, UK) was added in the Seeding or Maintenance Media until day 1 or day 3, respectively.
Orr B., Sutton K., Christian S., Nash T., Niemann H., Hansen L.L., McGrew M.J., Jensen S.R, & Vervelde L. (2021). Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer. Veterinary Research, 52, 142.
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6

Cryopreservation and Resuscitation of Enteroids

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To cryopreserve the enteroids the growth medium was removed and 1 mL ice-cold advanced DMEM/F12 medium containing 1X B27 supplement minus vitamin A, 25 μg/mL gentamicin and 100 U/mL penicillin/streptomycin was added directly to the Matrigel plug. The resulting enteroid suspension was then pelleted by centrifugation at 290 × g for 5 min at 4 °C. The enteroids were then resuspended in Cryostor CS10 cryopreservation medium (STEMCELL Technologies) at 1000 enteroids/mL. Cryovials were stored overnight in a Mr Frosty freezing container (ThermoFisher Scientific) at −80 °C, and then transferred to −155 °C for long-term storage.
To resuscitate the enteroids, the cryovials were thawed in a water bath at 37 °C. The enteroid suspension was then transferred to a 15 mL falcon tube containing 2 mL advanced DMEM/F12 medium containing 1X B27 supplement minus vitamin A, 25 μg/mL gentamicin and 100 U/mL penicillin/streptomycin and 1% BSA. The cryovial and lid were also washed twice with 1 mL of the above medium, and added to the enteroid suspension. The enteroids were then pelleted by centrifugation at 290 × g for 5 min at 4 °C, and cultivated as above.
Hamilton C.A., Young R., Jayaraman S., Sehgal A., Paxton E., Thomson S., Katzer F., Hope J., Innes E., Morrison L.J, & Mabbott N.A. (2018). Development of in vitro enteroids derived from bovine small intestinal crypts. Veterinary Research, 49, 54.
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7

Isolation and Differentiation of Primary Human Immune Cells

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Buffy coats from healthy donors were purchased from the Stanford Blood Center under an IRB-exempt-protocol. Leukopaks from healthy donors were purchased from StemCell Technologies. Primary human T cells were purified by negative selection using the RosetteSep Human T cell Enrichment kit (StemCell Technologies) and SepMate-50 tubes. T cells were cryopreserved at 2 × 10 7 cells per ml in CryoStor CS10 cryopreservation medium (StemCell Technologies) until use. Primary peripheral monocytes were purified through successive density gradients using Ficoll (Sigma-Aldrich) and Percoll (GE Healthcare). Monocytes were then differentiated into macrophages by 7-9 days of culture in IMDM + 10% AB human serum (Life Technologies).
Yamada-Hunter S.A., Theruvath J., McIntosh B.J., Freitas K.A., Lin F., Radosevich M.T., Leruste A., Dhingra S., Martinez-Velez N., Xu P., Huang J., Delaidelli A., Desai M.H., Good Z., Polak R., May A., Labanieh L., Bjelajac J., Murty T., Ehlinger Z., Mount C.W., Chen Y., Heitzeneder S., Marjon K.D., Banuelos A., Khan O., Wasserman S.L., Spiegel J.Y., Fernandez-Pol S., Kuo C.J., Sorensen P.H., Monje M., Majzner R.G., Weissman I.L., Sahaf B., Sotillo E., Cochran J.R, & Mackall C.L. (2024). Engineered CD47 protects T cells for enhanced antitumour immunity. Nature, 630(8016).
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8

Autologous CAR T-cell Cytotoxicity Assay

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Whole blood was derived from the Stanford Blood Bank and either used directly for Flow Cytometric analysis following ACK lysis and FC-blocking (BD Biosciences) or for isolation of distinct cell populations. T cells were isolated as described above. Granulocytes and B-cells were isolated using a whole blood column kit (Miltenyi) and magnetic beads for granulocytes (StraightFrom Whole Blood CD66b MicroBeads, Miltenyi) and for B-cells (StraightFrom Whole Blood CD19 MicroBeads, Miltenyi). Autologous T cells were activated as described above. Granulocytes were viably cryopreserved in CryoStor, CS10 cryopreservation medium (Stem Cell Technologies) and until use. B-cells were cultured and expanded for 10 days in ExCellerate B Cell Media, Xeno-Free (R&D Systems) supplemented with the CellXVivo Human B Cell Expansion Kit (R&D Systems) and a bone marrow stroma feeder-layer using HS-5 cells. CAR T cells were transduced as described above. Co-cultures between 0.1×106 autologous CAR T cells and NB tumor cells (NBSD), freshly harvested B-cells and thawed granulocytes were performed on day 10 after T cell activation at 1:1 ratios between effector and target cells. After 24 hours, supernatant culture media was collected stored at −20 °C until analyzed by ELISA and remaining cells were analyzed by Flow Cytometry.
Heitzeneder S., Bosse K.R., Zhu Z., Zhelev D., Majzner R.G., Radosevich M.T., Dhingra S., Sotillo E., Buongervino S., Pascual-Pasto G., Garrigan E., Xu P., Huang J., Salzer B., Delaidelli A., Raman S., Cui H., Martinez B., Bornheimer S.J., Sahaf B., Alag A., Fetahu I.S., Hasselblatt M., Parker K.R., Anbunathan H., Hwang J., Huang M., Sakamoto K., Lacayo N.J., Klysz D.D., Theruvath J., Vilches-Moure J.G., Satpathy A.T., Chang H.Y., Lehner M., Taschner-Mandl S., Julien J.P., Sorensen P.H., Dimitrov D.S., Maris J.M, & Mackall C.L. (2021). GPC2-CAR T Cells Tuned for Low Antigen Density Mediate Potent Activity Against Neuroblastoma Without Toxicity.. Cancer cell, 40(1), 53-69.e9.
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