Optimization of Viable Glioblastoma Cryopreservation for Establishment of Primary Tumor Cell Cultures

November 12, 2020
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eaaci2015

Development of new media formulations for cell culture operations based on regression models

The paper discusses modelling and optimization of multi-component cell culture medium. The specific productivity (Qp) was considered a function of the medium components and possible interactions described by linear factors, two-way interactions and squared terms that results in a high dimensional problem where the number of variables p (represented by the medium components and their interactions) is much larger than the number of observations n.
Principal Components Regression (PCR), Partial Least Squares (PLS), Lasso and Elastic Net regressions were compared as modelling tools to deal with a high dimensional [Formula: see text] problem. PCR and PLS regression models resulted in better prediction results and were used for robust optimization of the medium composition by a nonlinear optimization. The case studies show that it is possible to formulate new media that result in higher Qp than the ones provided by the initial media experiments available. Also, the multivariate statistical approach permitted us to select media that is most informative about the optimum thus permitting modelling and optimization with a reduced set of initial experiments.

Heat Stress Triggers Differential Protein Accumulation in the Extracellular Matrix of Sorghum Cell Suspension Cultures

Plants reprogram gene expression as an adaptive response to survive high temperatures. While the identity and functions of intracellular heat stress-responsive proteins have been extensively studied, the heat response of proteins secreted to the extracellular matrix is unknown. Here, we used Sorghum bicolor, a species adapted for growth in hot climates, to investigate the extracellular heat-induced responses. When exposed to 40 C for 72 h, heat-sensitive Arabidopsis cell suspension cultures died, while ICSB338 sorghum cell cultures survived by activation of a transcriptional response characterized by the induction of Gentaur.
Quantitative proteomic analysis of proteins recovered from cell culture medium revealed specific heat stress-induced protein accumulation within the sorghum secretome. Of the 265 secreted proteins identified, 31 responded to heat (2-fold change), with 84% possessing a predicted signal peptide for targeting to the classical secretory pathway. The differentially accumulated proteins have putative functions in metabolism, detoxification, and protein modifications. A germin (SORBI_3003G427700) was highly heat-inducible at both protein and gene level. Overall, our study reveals new insights into sorghum responses to heat and provides a useful resource of extracellular proteins that could serve as targets for developing thermotolerant crops. Data are available via ProteomeXchange with identifier PXD021536.

Xenograft and cell culture models of Sézary syndrome reveal cell of origin diversity and subclonal heterogeneity

Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading.
These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.
eaaci2015

eaaci2015

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Optimization of Viable Glioblastoma Cryopreservation for Establishment of Primary Tumor Cell Cultures

 

Background: Technologies related to the establishment of primary tumor cell cultures from solid tumors, including glioblastoma, are increasingly important to oncology research and practice. However, processing of fresh tumor specimens for establishment of primary cultures on the day of surgical collection is logistically difficult. The feasibility of viable cryopreservation of glioblastoma specimens, allowing for primary culture establishment weeks to months after surgical tumor collection and freezing, was demonstrated by Mullins et al. in 2013, with a success rate of 59% that was not significantly lower than that achieved with fresh tumor tissue. However, research targeting optimization of viable glioblastoma cryopreservation protocols for establishment of primary tumor cultures has been limited.
Objectives: The objective of this study was to optimize glioblastoma cryopreservation methods for viable cryobanking and to determine if two-dimensional (2D) or three-dimensional (3D) culture conditions were more supportive of glioblastoma growth after thawing of frozen tumor specimens.
Methods: Portions of eight human glioblastoma specimens were cryopreserved by four different protocols differing in the time of enzymatic digestion (before or after cryopreservation), and in the type of cryopreservation media (CryoStor CS10 or 10% dimethyl sulfoxide and 90% fetal calf serum). After 1 month, frozen tissues were thawed, enzymatically digested, if not digested before, and used for initiation of 2D or 3D primary tumor cultures to determine viability.
Results: Among the tested cryopreservation and culturing protocols, the most efficient combinations of cryopreservation and culture were those associated with the use of CryoStor CS10 cryopreservation medium, enzymatic digestion before freezing, and 2D culturing after thawing with a successful culture rate of 8 out of 8 cases (100%). Two-dimensional cultures were in general more efficient for the support of tumor cell growth after thawing than 3D cultures.

Metformin partially reverses the inhibitory effect of co-culture with ER-/PR-/HER2+ breast cancer cells on biomarkers of monocyte antitumor activity

Background: Immune activities of monocytes (MOs) can be altered within the microenvironment of solid malignancies, including breast cancer. Metformin (1,1-dimethylbiguanide hydrochloride, MET), has been shown to decrease tumor cell proliferation, but its effects have yet to be explored with respect to MOs (monocytes) activity during their crosstalk with breast cancer cells. Here, we investigated the effects of MET on overall phenotypic functional activities, including cellular immunometabolism and protective redox signaling based-biomarkers, intracellular free calcium ions (ifCa2+), phagocytosis and co-operative cytokines (IFN-γ and IL-10) of autologous MOs before and during their interplay with primary ER-/PR-/HER2+ breast cancer cells.
Methods: Human primary breast cancer cells were either cultured alone or co-cultured with autologous MOs before treatment with MET.
Results: MET downregulated breast cancer cell proliferation and phagocytosis, while having no significant effect on the ratio of phosphorylated Akt (p-Akt) to total Akt. Additionally, we observed that, in the absence of MET treatment, the levels of lactate dehydrogenase (LDH)-based cytotoxicity, catalase, ifCa2+, IL-10 and arginase activity were significantly reduced in co-cultures compared to levels in MOs cultured alone whereas levels of inducible nitric oxide synthase (iNOS) activity were significantly increased. In contrast, MET treatment reduced the effects measured in co-culture on the levels of LDH-based cytotoxicity, arginase activity, catalase, ifCa2+, and IFN-γ. MET also induced upregulation of both iNOS and arginase in MO cells, although the increase did not reach significant difference for iNOS activity.
Moreover, MET induced a robust increase of superoxide dismutase (SOD) activity in MOs, but not in MOs co-cultured with breast cancer cells. Furthermore, MET markedly upregulated the levels of IFN-γ production and downregulated those of IL-10 in isolated MOs, while inducing a slight opposing up-regulation of IL-10 production in co-cultures.
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Description: Cryopreserved vial (10 x 10^6 cells) of CD8+ T cells that were negatively selected from freshly isolated primary human peripheral blood mononuclear cells (PBMCs). The PBMCs came from a healthy donor, and were isolated from whole blood or leukapheresis samples using a Ficoll gradient. Magnetic antibodies to monocytes, granulocytes, CD4+ T cells, gamma/delta T cells and other immune subsets present in PBMCs were then used to purify untouched CD8+ T cells via immunomagnetic separation. Before and after CD8+ T cell isolation, the cells were stained to evaluate purity and viability by flow cytometry. Cells were cryopreserved in CryoStor CS10 cryopreservation medium (Stemcell, #07930) at a controlled rate._x000D_Source
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Thymocyte Expressed, Positive Selection Associated 1 (TESPA1) Antibody
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Description: Human neural progenitor cells (PSA-NCAM+) are derived from whole brain that are quickly dissociated into single cells and cultured. Human PSA-NCAM+ neuronal cells are from a single donor and purified using magnetic cell separation. These cells are greater than 90 % pure as assessed by flow cytometry. Human PSA-NCAM+ neuronal cells enable researchers to produce various sensory neurons, motor neurons, and their precursors found in the central nervous system. Human PSA-NCAM+ neuronal cells may be used for various types of in vitro, in vivo, or transplantation studies into normal or diseased systems. These cells are a great source for your neural-toxicity screening assays.Development period: Prenata
Thymocyte Expressed, Positive Selection Associated 1 (TESPA1) Antibody (Biotin)
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4GB USB memory stick - EACH
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CD45RA/CD45RO/CD3/CD4
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ICP-MS Memory Test 1 - 125ML
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EUR 544.05
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CL-MEM-2 125ML
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Recombinant Saccharomyces cerevisiae Prospore formation at selected spindle poles protein 1 (PFS1)
MBS1059505-002mgBaculovirus 0.02mg(Baculovirus)
EUR 1120
Recombinant Saccharomyces cerevisiae Prospore formation at selected spindle poles protein 1 (PFS1)
MBS1059505-002mgEColi 0.02mg(E-Coli)
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Recombinant Saccharomyces cerevisiae Prospore formation at selected spindle poles protein 1 (PFS1)
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MBS1059505-01mgEColi 0.1mg(E-Coli)
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MBS1059505-01mgYeast 0.1mg(Yeast)
EUR 1065
Human CD4+/CD45RA+/CD25-Naïve T Cells
ABC-TC4003 1 vial Ask for price
Description: First, NPB-CD4+ T Cells are negatively isolated using immunomagnetic CD4+ isolation kit from mononuclear cells. Next, CD45RO MicroBeads are used to deplete the CD45RO+ cell population, leaving an untouched, purified CD4+/CD45RA+ Naïve T Cell population.
CD19 Positive Cell Isolation Kit
78564-1 1 x 10^8 Cells
EUR 70
Description: The CD19 Positive Cell Isolation Kit is designed to magnetically separate CD19-expressing-cells from a complex immune cell population. This kit is optimized for the isolation of CD19 positive cells from normal human peripheral blood mononuclear cells (PBMCs). Cells are incubated with the antibody:bead complex and placed on a magnet for quick and easy separation. When placed on the magnet, CD19-positive cells will be immobilized along the side of the tube while undesired CD19-negative cells will remain in suspension for easy removal.
CD19 Positive Cell Isolation Kit
78564-2 1 x 10^9 Cells
EUR 630
Description: The CD19 Positive Cell Isolation Kit is designed to magnetically separate CD19-expressing-cells from a complex immune cell population. This kit is optimized for the isolation of CD19 positive cells from normal human peripheral blood mononuclear cells (PBMCs). Cells are incubated with the antibody:bead complex and placed on a magnet for quick and easy separation. When placed on the magnet, CD19-positive cells will be immobilized along the side of the tube while undesired CD19-negative cells will remain in suspension for easy removal.
Porcine circovirus 2 (PCV2) Capsid Insect Cells Cell Lysate (WB positive control)
MBS8116150-03mg 0.3mg
EUR 280
Porcine circovirus 2 (PCV2) Capsid Insect Cells Cell Lysate (WB positive control)
MBS8116150-5x03mg 5x0.3mg
EUR 1205
TESPA1 (Myc-DDK tagged) - Homo sapiens thymocyte expressed, positive selection associated 1 (TESPA1), transcript variant 4
RC233938 10 µg Ask for price
TESPA1 (Myc-DDK tagged) - Homo sapiens thymocyte expressed, positive selection associated 1 (TESPA1), transcript variant 3
RC233939 10 µg Ask for price
TESPA1 (untagged) - Homo sapiens thymocyte expressed, positive selection associated 1 (TESPA1), transcript variant 3
SC333287 10 µg Ask for price
TESPA1 (untagged) - Homo sapiens thymocyte expressed, positive selection associated 1 (TESPA1), transcript variant 4
SC332803 10 µg Ask for price
TESPA1 (GFP-tagged) - Homo sapiens thymocyte expressed, positive selection associated 1 (TESPA1), transcript variant 4
RG233938 10 µg Ask for price
TESPA1 (GFP-tagged) - Homo sapiens thymocyte expressed, positive selection associated 1 (TESPA1), transcript variant 3
RG233939 10 µg Ask for price
CD4+ T Cells-Lupus
ABC-SC0154T 1 vial Ask for price
Description: SLE CD4+ T Cells are available from Gentaur. All are listed on the certificate of analysis provided with each lot.
CD45RO, T-cell
1107NF-083D 5ml
EUR 180
Human Cord Blood CD4+/ CD45RA+ Naive T Cells
CBCD4-45RA-C10M 10 million
EUR 1378.8
Human Cord Blood CD4+/ CD45RA+ Naive T Cells
CBCD4-45RA-C15M 15 million
EUR 1678.8
Human Cord Blood CD4+/ CD45RA+ Naive T Cells
CBCD4-45RA-C5M 5 million
EUR 838.8
Human Cord Blood CD4+/ CD45RA+ Naive T Cells
CBCD4-45RA-F10M 10 million
EUR 1798.8
Human Cord Blood CD4+/ CD45RA+ Naive T Cells
CBCD4-45RA-F15M 15 million
EUR 1980
Human Cord Blood CD4+/ CD45RA+ Naive T Cells
CBCD4-45RA-F5M 5 million
EUR 1198.8
Human Cord Blood CD4+/CD45RA+ Naïve T Cells
ABC-TC3377 1 vial Ask for price
Description: First, Cord Blood-CD4+ T Cells are negatively isolated using an indirect immunomagnetic CD4+ labeling system from mononuclear cells. Next, CD45RO MicroBeads are used to deplete the CD45RO+ population, leaving a purified CD4+/CD45RA+ Naïve T Cell population.
Influenza A H1N1 (A/Bel/1942) Hemagglutinin/HA Insect Cells Cell Lysate (WB positive control)
MBS8116113-03mg 0.3mg
EUR 280
Influenza A H1N1 (A/Bel/1942) Hemagglutinin/HA Insect Cells Cell Lysate (WB positive control)
MBS8116113-5x03mg 5x0.3mg
EUR 1205
Coxsackievirus A16 (Cox A16) (strain G-10) VP1 Insect Cells Cell Lysate (WB positive control)
MBS8116137-03mg 0.3mg
EUR 280
Coxsackievirus A16 (Cox A16) (strain G-10) VP1 Insect Cells Cell Lysate (WB positive control)
MBS8116137-5x03mg 5x0.3mg
EUR 1205
Coxsackievirus A16 (Cox A16) (strain G-10) VP4 Insect Cells Cell Lysate (WB positive control)
MBS8116138-03mg 0.3mg
EUR 280
Coxsackievirus A16 (Cox A16) (strain G-10) VP4 Insect Cells Cell Lysate (WB positive control)
MBS8116138-5x03mg 5x0.3mg
EUR 1205
Enterovirus D68 (EV-D68) (strain Fermon) VP1 Insect Cells Cell Lysate (WB positive control)
MBS8116073-03mg 0.3mg
EUR 280
Enterovirus D68 (EV-D68) (strain Fermon) VP1 Insect Cells Cell Lysate (WB positive control)
MBS8116073-5x03mg 5x0.3mg
EUR 1205
BioMag® SelectaPure Human CD4+ T cell Enrichment System
85074-1 1ml
EUR 681.48
BioMag® SelectaPure Human CD4+ T cell Enrichment System
85074-5 5ml
EUR 2661.12
Zika virus (ZIKV) (strain Zika SPH2015) ZIKV-NS1 Insect Cells Cell Lysate (WB positive control)
MBS8116148-03mg 0.3mg
EUR 280
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