ComfortFreezer™: A Benchtop LN2–Free Programmable Freezer for Cryopreservation of Adherent Cells in Multi-Well Plates for Cell-Based High Content Screening

2012 ◽  
Author(s):  
Matthew D. Bockman ◽  
Igor I. Katkov ◽  
Stephen B. Jones ◽  
Vsevolod Katkov ◽  
Ilya Yakhnenko
Keyword(s):  
Pluripotent Stem Cells ◽  
High Content Screening ◽  
Toxicity Tests ◽  
Neural Cells ◽  
Adherent Cells ◽  
Environmental Toxicity ◽  
Toxicity Evaluation ◽  
Drug Candidates ◽  
Large Numbers ◽  
Dissociated Cells

Human pluripotent stem cells (hPSCs) and their progeny such as hPSC-derived cardiomyocytes and neural cells hold great potential as a source for cell therapy and regenerative medicine, as well can be effectively used for high high content screening (HCS) of drug candidates and for toxicity tests. Cryopreservation (CP), storage, and shipment of the cells are key elements for eventual clinical, pharmaceutical and environmental applications, which will require large numbers of quality controlled and ready for use cells. Traditionally, the cells are frozen in suspensions of either fully dissociated cells) or loosely associated clusters such as clumps of hPSCs, clusters of beaters”of cardiomyocytes, (“or neurospheres of neural precursors. Beside logistical inconvenience for some applications such as HCS, additional manipulation with the cells (detachment, dissociation and centrifugation) can introduce substantial stress to the cells prior to freezing and after thawing, which per se may tremendously decrease the cell cryosurvival and functionality. Here, we are presenting ComfortFreezer™, a novel bench-top device specifically designed for cryopreservation in multi-well plates for cell-based high content screening (HCS), which combines a liquid-nitrogen (LN2) free programmable freezer This cryogenic equipemnt can bring serious advantage for HCS in drug screening, environmental toxicity evaluation, and other variety of HCS-based applications.

scholarly journals DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Igor I. Katkov ◽  
Natalia G. Kan ◽  
Flavio Cimadamore ◽  
Brandon Nelson ◽  
Evan Y. Snyder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ethylene Glycol ◽  
Pluripotent Stem Cells ◽  
Toxicity Tests ◽  
Adherent Cells ◽  
Freezing And Thawing ◽  
Rock Inhibitor ◽  
Commercial Applications ◽  
Induced Pluripotent ◽  
Human Ipsc

Three modes for cryopreservation (CP) of human iPSC cells have been compared:STD: standard CP of small clumps with 10% of CPA in cryovials,ACC: dissociation of the cells with Accutase and freezing in cryovials, andPLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells inACCandPLTwere frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI.Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze®concept developed by CELLTRONIX). CP of cells directly in plates inready-to-goafter thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests.

scholarly journals Cell-Based Assay Design for High-Content Screening of Drug Candidates

2016 ◽  
Vol 26 (2) ◽  
pp. 213-225 ◽  
Author(s):  
Gregory Nierode ◽  
Paul S. Kwon ◽  
Jonathan S. Dordick ◽  
Seok-Joon Kwon
Keyword(s):  
High Content Screening ◽  
Drug Candidates ◽  
Assay Design

Abstract 20699: GMP-Grade Cardiomyocyte Differentiation Media System for Pluripotent Stem Cells in Basic and Translational Research

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Shayne Boucher ◽  
Stacy Jones ◽  
David Kuninger ◽  
Mohan Vemuri
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Translational Research ◽  
Pluripotent Stem Cells ◽  
Troponin T ◽  
Fold Increase ◽  
Cell Number ◽  
Cardiomyocyte Differentiation ◽  
Media System ◽  
Large Numbers

Objective: Current protocols for differentiating pluripotent stem cells (PSCs) have led to heterogeneous results, varying purity levels, and long lead times for generation of cardiomyocytes. We hypothesized that a simplified and rapid cardiomyocyte differentiation media system can be developed in a scalable workflow to enable generation of large numbers of consistent, spontaneously active cardiomyocytes that could be used in basic and translational research. Methods: High quality PSCs were maintained under xenofree, feeder-free culture conditions. At time of passaging, PSC were dissociated with 0.5 mM EDTA, seeded on 1:100 Geltrex © -coated surface as small clusters at ~0.5 to 1 x 10 5 /well of a 12-well plate and maintained for four days under serum-free condition. After reaching target confluence of ~60 to 80%, an induction media was added for two days followed by addition of a second induction media for two days. After the induction step, the media was replaced with maintenance media and re-fed every other day for up to five weeks. PSC-derived cardiomyocytes were analyzed by morphology, gene expression, flow cytometry, immunocytochemistry and multi-electrode array (MEA). Results: We observed individual beating cells by Day 7 and contracting syncytia by Day 10. An over 100 fold increase in cell number was noted from the time of plating to generation of contracting syncytia of cardiomyocytes. Quantitative flow cytometry detected populations of troponin T type 2 (TNNT2)-immunoreactive cells that reached as high as 96.6%. Number of TNNT2-positive cells dropped by 20% when induced at 90% versus 60% confluency. PCR studies confirmed expression of mesoderm (T, MIXL1, MESP1), cardiac mesoderm (ISL1, GATA4, MEF2C) and mature cardiomyocyte genes (NKX2.5, TNNT2, MYH6). Immunocytochemistry studies verified expression of cardiac markers NKX2.5,GATA4, MEF2C, TNNT2 and MYH6. Initial MEA studies corroborated the presence of electrically active cells. Conclusions: We conclude that a simplified complete differentiation media system could serve as a standardized culture system for generating large numbers of consistent, spontaneously active cardiomyocytes for basic and translational research studies.

scholarly journals Background Level of Pops in Ground Water Assessed on Chemical and Toxicity Analysis of Exposed Semipermeable Membrane Devices

2009 ◽  
Vol 2 ◽  
pp. ASWR.S2128 ◽  
Author(s):  
Vladimír Kočí ◽  
Tomáš Ocelka ◽  
Roman Grabic
Keyword(s):  
Sampling Site ◽  
Detection Limits ◽  
Semipermeable Membrane ◽  
Toxicity Tests ◽  
General Level ◽  
Contamination Level ◽  
Toxicity Evaluation ◽  
Semipermeable Membrane Devices ◽  
Secondary Contamination

Persistent compounds are present around almost the entire world. The level of contamination in very old groundwater sources (Cennoman bedrock Mesozoic, approximately 100 millions year old) was assessed. This offers an information about realistic natural background. Together with chemical analysis a toxicity evaluation of sampled sites was performed. Semipermeable membrane devices were applied as a sampling system. Exposed SPMDs were analyzed both for chemical contain of POPs and toxicity properties. The chemical analyses of PAHs were made by HPLC-FLD, PCBs and OCPs were analysed by GC/MS/MS on GCQ or PolarisQ (Thermoquest). Toxicity bioassays on alga Desmodesmus subspicatus, bacteria Vibrio fischeri and crustacean Daphnia magna was performed. The results show very low contamination of groundwater with POPs with concentrations close to detection limits of applied analytical tools. Even this low contamination was possible to rank based on the obtained toxicity data. Toxicity proved to be a good parameter for determination of relative POPs contamination where concentration is near to detection limits and thus correct determination of all POPs cannot be undertaken. Although contamination levels were found to be very low, a secondary contamination of PCBs through the bedrock was observed. Organochlorine pesticides were found at a sampling site near a mouth of the ground watershed. Applied toxicity tests confirmed the presence of toxic substances and marked sites of higher contamination. Application of toxicological parameter Vtox allowed the ranking of assessed sites by their contamination level even in cases where concentrations of pollutants were near or under detection limits and it was not therefore possible to rank the sites on the basis of chemical parameters. Toxicity response of bioassays obtained on SPMDs exposed in clean groundwater can be used as a background toxicity values for further SPMD applications. Secondary contamination with PCBs and pesticides was detected in Cennoman groundwater. Toxicity evaluation of SPMD extract can be used as an effective tool for ranking of general level of water contamination.

344 LOSS OF CONSTRAINTS IMPOSED BY TISSUE ENVIRONMENT ACTIVATES EARLY SIGNALS OF CELLULAR REPROGRAMMING

2015 ◽  
Vol 27 (1) ◽  
pp. 260
Author(s):  
D. A. Anzalone ◽  
D. Iuso ◽  
P. Toschi ◽  
F. Zacchini ◽  
G. E. Ptak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Adherent Cells ◽  
Pluripotency Factors ◽  
Differentiated Cells ◽  
Nanog Expression ◽  
Muse Cells

Pluripotency is the ability of one cell to generate every cell type of the 3 germ layers, a property typically owned by embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), with some exceptions; multilineage-differentiating stress-enduring (Muse) cells are an example. Muse cells, described as pre-existing pluripotent stem cells in mesenchymal tissues (Kuroda et al. 2010) are able to form clusters from single cells in suspension culture, express pluripotency factors and differentiate into cell types of the 3 germ layers, like ESC and iPSC. In addition, Muse cells are proposed to be the only source of cells capable to generate iPSC by current methodologies (Wakao et al. 2011). However, it is unclear whether they are normally present in adult tissue, derive from precursors stem or differentiated cells, or are induced by the in vitro conditions. In our work, we tested the hypothesis that the transition from a committed (tissue) to an uncommitted (in vitro culture) environment triggers in the cells the activation of a default gene circuitry leading to pluripotency. Adult skin fibroblasts were obtained from sheep ear biopsy (n = 3) and expanded in vitro (A) or cultured in suspension in hanging drops (B) or in nonadherent dishes (C) in MEM with 10% FBS. In a subsequent experiment, clonal expansion was attempted by culturing single suspension cells in drops of medium (D). Pluripotency was assessed analysing Oct4 and Nanog expression, using real-time PCR (mRNA) and Western blotting (protein), in cultured fibroblasts compared to whole ear biopsy (30-day-old fetus was used as positive control, CTR). Furthermore, in adherent cells (A) and in clusters obtained from suspension culture (B, C, D), Oct4 and Nanog expression was compared by immunofluorescence. We found that while in the ear biopsy not one of these pluripotency markers was expressed, in in vitro-expanded fibroblasts both mRNA and protein expression was detected; mRNA expression value (mean ± s.e.m. relative to CTR) was 0.59 ± 0.18 for Nanog and 0.2 ± 0.07 for Oct4. Moreover, fibroblasts in suspension (B, C, D) were able to form clusters [obtained from 32% (16/50) of single cells, D] similar to those normally obtained with ESC, iPSC. and Muse cells. All the clusters (B, C, D) showed a more intensive signal of Oct4 and Nanog protein compared to adherent cells by immunofluorescence. In the present work we demonstrate that adult somatic cells (skin fibroblasts) express key pluripotency factors, such as OCT4 and Nanog, in both adherent and suspension culture, after removal from the tissue (ear). We can conclude that the simple in vitro culture switches on the expression of pluripotency markers in adult somatic cells. Removal from the context of the tissue probably leads the cells to lose their tissue-specific identity and acquire a new undifferentiated one, which in an optimal condition culture could result in pluripotency. Our interpretation is that reprogramming must be an automatic, default response when differentiated cells are removed from the constraints imposed by a multicellular environment.

In vitro and in vivo toxicity evaluation of non-neuroleptic phenothiazines, antitubercular drug candidates

2019 ◽  
Vol 109 ◽  
pp. 104508 ◽  
Author(s):  
Sumayah Salie ◽  
Antoinette Labuschagné ◽  
Avril Walters ◽  
Sohair Geyer ◽  
Anwar Jardine ◽  
...  
Keyword(s):  
Antitubercular Drug ◽  
Toxicity Evaluation ◽  
In Vivo Toxicity ◽  
Drug Candidates

scholarly journals THE RELATIVE EXTENSIBILITY OF CELL SURFACES

1963 ◽  
Vol 17 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Murray D. Rosenberg
Keyword(s):  
Cell Culture ◽  
Mechanical Deformation ◽  
Culture Chamber ◽  
Cell Surfaces ◽  
Statistical Index ◽  
Degree Of Deformation ◽  
Large Numbers ◽  
Dissociated Cells ◽  
Definition Of

Observations have been made on the response, in vitro, of cultured and freshly dissociated cells to mechanical deformation. Large numbers of individual cells were studied by means of a special culture chamber bounded by two parallel glass coverslips whose spacing could be reduced from 140 to 2 microns in steps of roughly 0.5 micron. The degree of deformation required for herniation of the cell surface was measured. These measurements lead to the definition of a statistical index characteristic of the extensibility of cell surfaces. This index has been shown to be distinctive for several types of cells; to alter with certain stages of embryonic development; and to be stable with respect to the culturing of cells and certain alterations in the method of cell culture.

Sign in / Sign up

Export Citation Format

Share Document