Module 1: Cell Culture and Cell Phenotyping
The goal of the Cell Culture and Cell Phenotyping Module is to streamline cell culture and phenotyping analyses for vision researchers. The Director and staff of this Module provide training, technical support, and maintenance of key equipment to facilitate scientifically rigorous, cutting edge, vision research.
The Cell Culture arm of this module provides technical expertise to investigators to assist in the establishment of primary cultures and development of new immortalized cell lines. This first arm provides the following essential services:
1) Routine maintenance of immortalized cell lines
2) Maintenance of cryopreserved stocks of cell lines
3) Establishment of primary corneal and retinal cell lines
4) Routine screening for mycoplasma and endotoxin contamination
5) Short tandem repeat DNA profiling for cell line authentication
The Cell Phenotyping arm of this module is designed to provide technical expertise and assistance with ongoing cell phenotyping experiments. This includes overseeing the maintenance of all major pieces of equipment, training of investigators, students, postdocs, and staff on proper use, and guidance on experimental design and data analysis, including:
1) Flow cytometry
2) Cell sorting
3) High throughput cellular analysis
4) Live cell metabolic profiling
5) Microvesicle quantification and size determination
Collectively, both arms of this Module add scientific value to the work of vision scientists by reducing the time and effort required for basic cell culture assays such as viability and cell cycle assessments. The incorporation of a flow cytometer and a cell sorter also reduces the cost associated with recurrent use of the main flow cytometry facility on campus. Moreover, inclusion of a high throughput imaging cytometer allows for many of these same comparisons to be made without the need for trypsinization and extensive cell processing. Inclusion of tunable resistance pulse sensing for the real time analysis of cellular microvesicles adds an additional layer of innovation.
The Cell Culture and Cell Phenotyping (CCCP) laboratories are located within the Department of Ophthalmology on the 7th floor of the Florence (E) Building. The CCCP Module component units are all within close proximity to the other laboratories in the Department of Ophthalmology. They are housed in four rooms totaling 2,319 ft2. All laboratories are less than a 2 minute walk from each other, and no more than a 10-minute walk from the labs of the furthest UTSW Core User. The available equipment and resources are as follows:
- Cell Culture (E7.232B, 161 ft2 and E7.232, 750 ft2)
Room E7.232B has the following equipment that is relevant to the day-to-day function of this module: two laminar flow hoods; two CO2 incubators; two tri-gas incubators with oxygen regulation; one inverted compound microscope; and one heated water bath containing Lab Armor beads. Immediately adjacent, is room E7.232 which contains one refrigerated centrifuge; one refrigerator/ freezer dedicated to cell culture activities; and a Nexcelom Cellometer for rapid determination of cell number, cell viability, transfection efficiency, and cell cycle assays.
- Flow Cytometry/Cell Sorting (E7.134, 288 ft2)
Equipment in this laboratory includes an Attune NxT Flow Cytometer from Life Technologies and a Sony SH800 Cell Sorter. The flow cytometer is equipped with multiple lasers for analysis of multi-fluorescent labelled cell populations and acoustical focusing for improved cell separations. The cell sorter has 4 lasers providing flexibility for experiments with up to 6 colors and with multiple sort collection formats.
- Live Cell Metabolic Phenotyping (In the 1120 ft2 Common Equipment Lab, E7.212)
This Module component contains an Agilent Seahorse XFp live cell metabolic analyzer. In addition, there is a standard, non-CO2 incubator that is used for metabolic studies.
- High Throughput Cellular Analysis (also in E7.212)
The Department of Ophthalmology owns two pieces of equipment that are useful for high throughput analysis. The first is a BioTek Synergy 2 Multi-Mode Microplate Reader with detection modes for fluorescence, luminescence and UV-visible absorbance. It is equipped with Gen5 Reader Control and Data Analysis Software. The second is a recently purchased Celigo Imaging Cytometer from Nexcelom. The Celigo is a bench-top, high throughput, micro-well plate based, bright field and fluorescent imaging system. It consists of 4 fluorescent channels (UV to far red) and has extensive applications including bright field cell counting, wound-healing and cell growth assays, cell line development, induced pluripotent stem cell (iPSC) reprogramming, 3D invasion and migration modeling, immune-oncology, virology, and fluorescent-based assays.
- Tunable Resistive Pulse Sensing (E7.232, 750 ft2)
This laboratory contains a qNANO Gold tunable resistive pulse sensor (TRPS) from IZON Sciences. The qNANO provides measurements of individual nanoparticle size at sub-nanometer precision, simultaneous measurements of size and concentration, and surface charge (zeta potential). This device is ideal for studies on exosomes and microvesicles, which currently represent a hot area of vision research.
- Routine Maintenance of Cell Cultures
Cell cultures are monitored quarterly for mycoplasma and endotoxin contamination using commercial testing kits. Backup stocks of cell cultures are stored in one of six liquid nitrogen storage tanks that are housed in our common equipment facility (E7.238). Backup stocks are re-derived every six months. In addition, to enhance the dissemination of these materials to foster scientific advancement within the vision science community, we routinely ship cell lines to all institutions within the United States and abroad, upon request.
- Primary Cell Cultures and Development of Ocular Cell Lines
A number of the Core PIs have extensive experience in developing primary cell cultures from human and murine ocular tissues. These include human retinal pigment epithelial cells, murine iris/ciliary body cells, human and murine corneal epithelial cells, human, rabbit and murine keratocytes, and human and murine corneal endothelial cells. Ocular cell lines established from various mutant, transgenic, and knockout mice have enormous potential for a wide variety of experiments. The ability to streamline cell line maintenance, cell authentication, and the establishment of new cell lines within one central facility provides a valuable resource to all investigators. The Module director, along with several of the other Core investigators, have extensive experience in developing immortalized ocular cell lines from primary cultures using viral vectors.
- Mycoplasma and Endotoxin Screening
Some of the Core investigators may choose to maintain their cell cultures within their own laboratory, but still utilize the Module’s screening service for detecting contamination. For these investigators, we offer ad hoc screening for mycoplasma and endotoxin. The value of a centralized screening service for cell culture contaminants is twofold. First, it frees PIs from having to continuously test for contaminants in their cell cultures, promoting more frequent cell culture screening. Second, it provides a consistent level of standardization for culture models among all PIs, increasing the rigor and reproducibility of their work. Commercial kits are used to detect contaminants. Contaminated cells are discarded and replaced with frozen stocks. Valuable cells that are contaminated with mycoplasma and which do not have backup frozen stocks are treated with Plasmocin (InvivoGen) to purge mycoplasma. In our hands, this has been an effective, albeit time-consuming, method for eliminating mycoplasma.
- Short Tandem Repeat (STR) DNA Profiling for Cell Line Authentication
In addition to mycoplasma and endotoxin screening, this module offers an STR profiling service. STR profiling is done through the American Tissue Culture Collection (ATCC). For cell lines maintained in this Facility, routine STR profiling is performed on all actively growing cultures every six months. Regular STR profiling is essential to monitor for cross contamination, genetic drift, and ensures accuracy in data collection. Cultures are tested prior to being frozen down for long-term storage. For cell lines maintained in individual investigator laboratories, the STR profiling service is available upon request. All STR profiling results is tracked in an established log. Determination of population doubling times to validate growth rate is done every 6 months using the Celigo Imaging Cytometer. Any change in doubling time or cell behavior necessitates re-authentication.
- Flow Cytometry and Cell Sorting
This service is designed to provide training and technical support for investigators to perform flow cytometry and cell sorting assays. This service includes an Attune NxT acoustic focusing cytometer that utilizes hydrodynamics focusing technology and advanced fluidics to analyze a wide range of samples with a sample flow rate of 12.5-1µl/min. The flow cytometer can be used for: 1) monitoring cell viability; 2) fluorescent protein analysis; 3) transcription factor studies; and, 4) immunophenotyping. The SONY SH800S bench top cell sorter utilizes 70-μm, 100-μm, and 130-μm microfluidic sorting chips to sort a variety of cell samples. The instrument allows sorting of cells in to tubes, 96 well and 384 well plates. The cell sorter can be used fir: 1) sorting and viability analysis of individual cells with a mixed population; 2) fluorescent protein analysis; and, 3) distinct resolution of individual cell populations using multicolor immunophenotyping assays.
- Metabolic Profiling
This service provides technical support for the analysis of metabolic profiling in live cells. This type of analysis has applications in all aspects of vision research. The Seahorse metabolic flux analyzer is able to measure metabolic rates within minutes in as few as 5000 cells per well. Outcome measures include characterization of metabolic phenotypes, mitochondrial function, glycolytic rates, ATP generation (including source of ATP generation: mitochondrial respiration versus glycolysis), and T-cell activation. The Seahorse XFp analyzer has an 8-well format that is designed to streamline measurements for low sample numbers and can be used with adherent or suspension cells, in addition to isolated mitochondria.
- High Throughput Cellular Analysis
This service is designed to streamline and reduce the time required for the quantitative assessment of key cellular functions. The major pieces of equipment within this service include a Bio-Tek Synergy 2 multi-mode plate reader and a Nexcelom Celigo Imaging Cytometer. The multi-mode plate reader measures absorbance values ranging from 200 nm to 999 nm and has fluorescence and luminescence capabilities. The Celigo Imaging Cytometer provides bright field imaging and quantitation in addition to 4 fluorescence channels. The Celigo is a powerful instrument that reduces the time needed for almost all functional cellular assays from days to minutes. The whole well cell counting feature is also essential for normalizing metabolic phenotyping data. The applications of the Celigo are extensive, well beyond basic phenotypic assays, and apply to all aspects of vision research, as discussed in the resource section.
- Tunable Resistive Pulse Sensing
The Department of Ophthalmology acquired a tunable resistive pulse-sensing (TRPS) unit for the direct measurement of nanoparticles. This service has been added to the CCCP Facility to provide size, concentration and surface charge analysis of exosomes and other microvesicles that are released from cells and present in body fluids. This service also provides training and technical assistance on the use of this device to measure nanoparticles of interest.