Module 1: Stem Cell, Organoid, and Cell Phenotyping
The goal of the Stem Cell, Organoid and Cell Phenotyping (SOCP) Module is to provide vision researchers with access to advanced technologies including stem cell and organoid development, extracellular vesicle analysis, and immunometabolism phenotyping. To accomplish this goal, this Module is structured into two arms. The first arm will support the development of induced pluripotent stem cells (iPSCs) and organoids relevant to the eye. The second arm will provide comprehensive services for immunometabolic profiling, including flow cytometry, high throughput cellular analysis, live cell metabolic profiling using Seahorse and high resolution respirometry (Oroboros), and analysis of extracellular vesicles from cell culture and human body fluids. Together, this Module will allow for the rigorous study of a wide range of NEI-focused research activities, including all NEI-funded research projects, and new collaborative and pilot projects. The director and staff of this Module will provide training, technical support, and maintenance of key equipment to facilitate scientifically rigorous and cutting-edge vision research.
The Stem Cell/Organoid arm of this module will provide the following essential services:
1) Technical support and reagents for the derivation, culture, validation, and cryopreservation of induced pluripotent stem cells (iPSCs)
2) Technical support for the generation and culture of organoids from human and mouse embryonic and adult stem cells
3) Reagents for mycoplasma testing, STR profiling, and karyotyping analysis
The Cell Phenotyping arm of this module will provide the following comprehensive analytical services:
1) Flow cytometry
2) Cell sorting
3) High throughput cellular analysis
4) Live cell metabolic profiling
5) Microvesicle quantification and size determination
Collectively, these services will provide our vision research community with state-of-the-art resources that will increase their experimental efficiency and support the generation of high quality, quantitative data for all aspects of visual science.
Facilities
The SOCP laboratories are located within the Department of Ophthalmology on the 7th floor of the Florence (E) Building. The SOCP 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 15 minutes from the labs of the furthest UTSW Core User.
Stem Cell Research Oversight Registration: Any investigators working with human embryonic stem cells and/or human induced pluripotent stem cells at UTSW are required to maintain an approved protocol on file with the UTSW Stem Cell Research Oversight Committee through the office of Research Support and Regulatory Management.
Equipment
Equipment in the SOCP is comprised of the following (separated by function):
- Stem cell and organoid culture
A 1300 Series A2 laminar flow hood equipped with a EVSXL Core Imaging System, a VWR CO2 incubator, a PHCbi multigas CO2/O2 controlled incubator containing two ThermoFisher rockers for organoid cultures, an Eppdendorf 5417R centrifuge, and a Sorvall ST8R centrifuge.
- Flow cytometry/cell sorting
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 programmed for multiple sort collection formats.
- Live cell metabolic phenotyping
An Agilent Seahorse XFp live cell metabolic analyzer and an Oroboros O2K high resolution respirometer that allows for the simultaneous measurement of oxidative phosphorylation and other metrics of mitochondrial metabolism. The O2K is equipped with both large and small volume chambers and a sample holder for whole tissue and adherent cell studies. In addition, there is a standard, non-CO2 incubator that is used for Seahorse sample preparation.
- High throughput cellular analysis
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 Module is also equipped with a Celigo Imaging Cytometer from Nexcelom. The Celigo is a bench-top, high throughput, micro-well plate based, brightfield and fluorescent imaging system. It consists of 4 fluorescent channels (UV to far red) and has extensive applications including brightfield 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.
- Extracellular vesicle analysis
A nanoparticle tracking analyzer (NTA, NanoSight NS300) for extracellular vesicle analysis. The NTA is configured with a 532 nm laser and a high sensitivity scientific CMOS camera. This allows for simultaneous measurement of vesicle size and concentration. Fluorescence is used for quantification of labeled vesicles. The NTA has additional applications in drug delivery, protein aggregation, and nanotechnology studies.
Services and Methodologies
- Derivation and validation of induced pluripotent stem cells (iPCSs)
This Module service will assist vision researchers with the derivation and culture of iPSCs. Somatic cells will be reprogrammed using the CytoTune-iPS 2.0 Sendai reprogramming (ThermoFisher). On average, 4 iPSC clones will be isolated and expanded into lines. iPSCs will be validated for 2 pluripotency markers (TRA01060 and SSEA4) using flow cytometry. Four clonal lines will be provided to the investigator with cryopreservation of 4-6 vials per clonal line. All cells will be tested for mycoplasma contamination prior to derivation. For investigators electing to generate iPSCs within their respective laboratory, training will be made available to their personnel. Each investigator will be required to submit their own Stem Cell Research Oversight Registration to the Office of Research Support and Regulatory Management at UTSW.
- Organoid development and culture
Human embryonic stem cells (hESCs, cell line WA09 – also known as H9, NIH approved collection) will be purchased from WiCell and used for the development of retinal and corneal organoids. Due to MTA requirements between WiCell and UTSW, each investigator will be required to purchase the cells directly from WiCell. Each investigator will also be required to submit their own Stem Cell Research Oversight Registration to the Office of Research Support and Regulatory Management at UTSW. Once hESCs are received, this Module will provide technical expertise and assist with cultures, as needed for each investigator. Since organoid development requires prolonged culture, this Module will provide the technical support that is required, streamlining organoid production for vision researchers. Investigators will be responsible for providing their own cell culture reagents. In addition, this Module will support new initiatives for vision researchers seeking to use mouse adult stem cells for organoids related to the visual system.
- Mycoplasma and endotoxin screening
Core investigators maintain their cell cultures within their own laboratories, but utilize the Module’s screening service for detecting contamination. For these investigators, we will provide commercialized kits for routine testing for mycoplasma and endotoxin. While contaminated cells are normally discarded and replaced with frozen stocks, for valuable cells that become contaminated, this Module will provide investigators with Plasmocin (InvivoGen) to purge mycoplasma. Making commercialized kits readily available to all investigators will provide a consistent level of standardization for culture models among all PIs and increase the rigor and reproducibility of their work.
- Short tandem repeat (STR) DNA profiling for cell line authentication
In addition to mycoplasma and endotoxin screening, this Module will offer an STR profiling service to be done through the American Tissue Culture Collection (ATCC). To accomplish this, the Module will stock sample collections kits for STR profiling for mouse and human cells. These test kits will be made readily available to all vision researchers on campus upon request. Regular STR profiling is essential to monitor for cross contamination, genetic drift, and ensures accuracy in data collection.
- Cytogenetic analysis
This Module service will offer karyotype analysis to investigators who need to confirm the absence of chromosomal abnormalities in their cell lines. This is particularly important with use of iPSCs and hESCs. All samples will be submitted directly to WiCell by either the investigator or Ms. Cao, with results normally returned within 7 – 10 days. Fees for this service will be covered by this Module for all vision researchers on campus.
- Flow cytometry and cell sorting
This Module 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. 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 into tubes, 96 well, and 384 well plates. The cell sorter can be used for: 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. In addition to training and technical support, this Module provides the necessary reagents for maintenance and use of both instruments.
- Metabolic profiling
This Module service will provide training for vision researchers in 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 measures oxygen consumption and extracellular acidification in real time, allowing for analysis of metabolic phenotypes, mitochondrial function, glycolytic rates, ATP-linked respiration, 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. The Oroboros O2K high resolution respirometer allows for the analysis of electron transport chain complexes, oxidative phosphorylation, coupling state, beta-oxidation of fatty acids, reactive oxygen species, ATP generation, mitochondrial membrane potential, and mitochondrial calcium levels in tissues, organoids, permeabilized cells, cell lysates, and isolated mitochondria.
- High throughput cellular analysis
This Module 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 brightfield 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 Seahorse metabolic phenotyping data. The applications of the Celigo are extensive, well beyond basic phenotypic assays, and apply to all aspects of vision research.
- Nanoparticle tracking analysis
The Department of Ophthalmology has a NanoSight NTA instrument for the direct analysis of nanoparticles. This equipment provides quantifiable measurements, such as size and concentration of exosomes and other microvesicles that are released from cells and present in body fluids. This Module will also provide training and technical expertise on the use of this device to measure other nanoparticles of interest.