Cell Preparation and Analysis Core
About the Core
The Cell Preparation and Analysis Core provides essential and emerging technologies in hematology and relevant training for junior investigators, thus considerably increasing research efficiency, reducing expense, and saving valuable investigator time to achieve better advancement of hematologic research.
In Vitro Hematopoiesis Assays
The Core will provide free training and cost-recovery-based services for in vitro assays of hematopoietic cells including liquid cultures for differentiation down specific hematopoietic lineages as well as clonogenic assays in semisolid medium. Clonogenic assays allow for growth of colony forming cells/units (CFU) and long term culture initiating cells (LTC-IC).
Core staff will teach YCCEH lab members how to perform the colony forming assays, as well as how to recognize the colony type by morphology. Additional training will include isolation of colonies for assay by cytospin and Wright-Giemsa staining. Such training will occur once per month, or as determined by the number of requests for training.
As a fee-based service, Core staff will perform colony assays, and return users with colony counts. Secondary or long-term culture can also be performed with increased fee structure. Colony analysis by cytospin and Wright-Geimsa staining will also be available as a service.
Automated Complete Blood Counts
Complete Blood Counts (CBC) are widely used in hematology. The Core uses a Hemavet veterinary hematology analyzer that provides accurate CBC counts from whole blood of mice, rats, dogs, and humans (research use only). Core staff will provide free training to teach individuals how to use the Hemavet independently, as well as the basics of interpreting the CBC results. Such training will occur once per month, or as determined by the number of requests for training. Users can use the Hemavet as needed for which they will be billed a charge based on cost recovery (current charge $16.00/sample).
Please contact Lin Wang at firstname.lastname@example.org for more information on these services.
Morphological assays of blood/bone marrow smears or tissue sections are important in hematology. The Core will synergize with the existing Yale Research Pathology Core and the Comparative Medicine Mouse Research Pathology Core to provide YCCEH members with comprehensive morphological analysis services.
Yale Research Pathology and Histology Core currently provides services on both human and mouse tissues, including hematopoietic tissues. The services (with cost-recovery) include preparation of samples, such as making tissue sections, as well as phenotype analysis together with requesting investigators. The YCCEH Morphologic Analysis service will provide expert consultation as well as training on specific assays for preparing hematological samples. Specifically, the Core will assist in analysis of bone marrow smear, blood smears, spleen sections, and cytospins analysis of cells grown in culture. Training will be provided for making blood and bone marrow smears; and 2) performing Wright-Geimsa staining. Such training will occur once per month, or as determined by the number of requests for training.
Contact Diane Krause for more information on these services.
Primary Human CD34+ Cells
Human CD34+ cells are widely utilized for modeling normal human hematopoiesis, either in vitro, or in vivo by transplantation into immunodeficient murine models. The Core will provide hematology researchers with purified, quality-controlled and cost-effective human CD34+ cell preparations, through a cost-recovery system.
Good Manufacturing Protocol (GMP) facility run by Dr. Krause for clinical cell therapy research, selection of CD34+ cells utilizes the Miltenyi CliniMacs device. Core staff will cryopreserve CD34+ selected cells at concentrations of 1, 5 and 20 x 106/ml in 10% DMSO in 1 ml aliquots. We also provide excess unprocessed G-CSF mobilized apheresis-derived peripheral blood mononuclear cells for research by maintaining an inventory of units that would otherwise be discarded. The cost of cells are $270.00 per million for Yale and $353.00 for external Non-Profits.
In addition, this core will also perform custom large-scale CD34 selections for investigators who need large cell numbers and/or who perform parallel investigations on the CD34- cells from the same donors.
Please contact Diane Krause for this special service.
Genetic Viral Library Service
Pooled genetic libraries are important approaches to unbiasedly identify functional genetic regulators in hematology. ShRNA, CRISPR and microRNA libraries have been frequently used to interrogate functional cellular pathways in pooled screens.
YCCEH will make available libraries produced by YCCEH researchers to the wider community. Currently, we are offering a microRNA overexpression library generated by the Lu laboratory at Yale. MicroRNAs are important regulators in hematology. Functional screens using this 461-miRNA containing library has been used successfully in both in vitro and in vivo functional screens to identify regulators in hematopoiesis. This library comes in two different versions, a MSCV-retrovirus based constitutive expression library and a lentivirus-based doxycycline-inducible library.
For shRNA and CRISPR libraries, due to intellectual property considerations, we are only providing service to produce virus for researchers once they obtain such libraries from other sources. Please refer to High titer virus services.
5ml MSCV-retrovirus constitutive miRNA library (providing pooled virus), cost: $300
200ul Concentrated Lentivirus inducible miRNA library (providing pooled virus), cost: $500
CRISPR Based Genomic Editing
CRISPR technology enables researchers to use customizable RNA guides to genetically modify the genome, control gene expression, or perform live cell imaging of specific genomic loci. CRISPR technology originates from an immune defense mechanism in prokaryotes. Engineering of the bacteria CRISPR system has led to much simplified molecular tools that can be readily applied in mammalian cells.
Currently, the most commonly used CRISPR system is based on that from Streptococcus pyogenes, composing primarily of two components: a protein named Cas9 (or its variants) as well as a small RNA named single guide RNA (sgRNA). Both components can be readily delivered to mammalian hematopoietic cells through either viral infection or plasmid transfection. In addition, for genomic engineering through homologous recombination, a third component of template DNA is needed, which can be delivered through transfection. Besides direct delivery into hematopoietic cells, CRISPR technology has been applied to quickly generate mouse models by delivering CRISPR components into single-cell embryos.
The following are possible with CRISPR technology:
- Genetic knockout through non-homologous end joining based random mutagenesis.
- Defined genomic alterations through homologous recombination
- Control gene activation or repression
- Live cell imaging of specific genomic loci
YCCEH provides service to advice experimental planning using CRISPR and provides CRISPR-related protocols upon request.
High Titer Virus Services
Retroviruses and its special subtype, lentiviruses, are essential tools to deliver genetic materials into mammalian hematopoietic cells. However, high titer viruses are required in many hematology applications, yet researchers are often troubled with the low titer of viruses that cannot be effectively used in their research applications. Due to this reason, YCCEH provides multiple services on high titer virus to the hematology community.
Advisory Service and Protocol Service:
YCCEH provides free service to advice experimental planning using viruses, including, but not limited to, choices of retro vs lenti virus, choices of vectors and safety considerations.
In addition, YCCEH provides for free proven protocols for high titer virus production upon request.
Production of High Titer Viruses:
YCCEH provides services to make viruses for researchers. The standard service involves generation of virus in 10 cm cell culture plates, yielding >15 ml of virus. Viruses will be packaged in 293T cells, using standard amphitropic packaging plasmids (i.e. with VSVG as envelop protein). Viruses will be filtered through 0.45 micron filter before delivering to researchers to remove any contaminating packaging cells.
Viral construct needs to be provided to YCCEH, with full disclosure of genetic composition due to safety considerations. Plasmid needs to be high quality midi or maxi prep product.
Cost: $75 per virus for the standard service. $75 additional for plasmid transformation and midi prep to be performed by YCCEH.
Additional services: see Concentrating Virus and Measuring Virus Titer
YCCEH provides services to concentrate viruses for researchers. This service is an add-on to the High Titer Virus Production service. We currently do not provide this service as a stand-alone service due to inability to guarantee viral performance.
Concentration step will be performed using high-speed centrifugation. We routinely achieve >20 fold enrichment of virus activity through this concentration step.
Cost: $50 for the first virus. $30 for each additional virus on the same order.
Measuring Viral Titer:
YCCEH provides services to measure virus titer for researchers. This service is an add-on to the High Titer Virus Production service or the Concentrating Virus service.
Viral titer will only be measured if there is a constitutive or doxycycline inducible fluorescence protein marker, or there is a constitutive or doxycycline inducible antibiotics selection marker in the viral construct.
Viral titer will be measured to reflect activity on BaF3 or K562 cells, or both. YCCEH will return the levels of transduction using 3 different viral amounts.
Cost: $60 for the first virus with fluorescent protein marker. $30 for each additional virus on the same order.
Cost: $50 for the first virus with fluorescent protein marker. $25 for each additional virus on the same order.
FACSorting and Analysis
FACS-based cell sorting is essential in hematopoietic research. The Core will synergize with the existing Yale Flow Cytometry Core to provide YCCEH members with comprehensive cell sorting services of human and mouse hematopoietic cells. In the Yale Flow Cytometry Core facility, 4 full-time personnel are available for performing sorting with four BD Aria machines, a Beckman Coulter MoFlo and a Sony SY3200. Core staff also train and/or assist individuals in performing their own sorts. The Flow Cytometry Core charges based on cost recovery, and costs are kept low due to the high volume of usage.
Complementary to the existing services by the Yale Flow Cytometry Core, the YCCEH Cell Preparation and Analysis Core will provide:
- assistance and advice in experimental design to YCCEH members, such as determining which subpopulation(s) may be optimal for a given experiment, which antibody combinations are most effective, which fluor combinations are best, which collection medium/reagent is most suitable, and whether double sorting may be necessary;
- training on performing population depletion, which is necessary to enrich for low-abundance populations before FACS-sorting; and
- hands-on training for antibody staining of hematopoietic cells.
Disease Specific iPSC Production
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) and then differentiating back to hematopoietic lineage is emerging as an important approach to model human hematopoietic development and non-malignant hematopoietic diseases. The Core will synergize with the existing Yale iPSC/ESC Core to provide YCCEH members with both iPSC generation service and iPSC/ESC to blood differentiation service (the latter to be mentioned in the next section).
Yale iPSC/ESC Core currently performs iPSC generation as a fee-for-service, which allows reprogramming differentiated cells into induced pluripotent cells using two non-integration techniques in defined media and feeder-free conditions. The technique can be either Sendai virus or episomal vectors. The source cells can be fibroblasts or peripheral blood mononuclear cells. This service includes delivery of at least 3 to 5 iPSC clones, each with 3 cryopreserved vials.
In addition to iPSC generation, the Yale iPSC/ESC Core also provides fee-based training for culturing iPSC/ESC cells, as well as assuring compliance with federal and non-federal regulations on ESC research.
The Cell Preparation and Analysis Core will complement these existing services by providing free consultation for YCCEH members, including advice in experimental design, considerations when using iPSC technology, as well as the technical feasibility and the advantages vs. limitations of such approaches.
GMP Grade Cell Processing
Good Manufacturing Practices (GMP) are necessary for preparing cells and viral reagents for clinical trials. The Cell Preparation and Analysis Core will synergize with the existing Yale Laboratory for Advanced Cell Therapy, a GMP laboratory run by Dr. Diane Krause, to help YCCEH members with GMP-grade cell preparation. Dr. Krause will provide free consultation and advice on this service. Charges will be based on cost-recovery.
The newly designed Yale Laboratory for Advanced Cell Therapy was completed in 2014, with 2 separate ISO 7 rooms, each fed by a class 10,000 shared equipment corridor. The laboratory can serve the cell therapy clinical trials at Yale University and other institutions. The laboratory staff has expertise in performing clinical-scale preclinical trials for FDA applications. To date, the laboratory has processed cells for seven investigator-initiated clinical trials at Yale as well as nine company-sponsored clinical cell therapy trials. As part of the U54 NIDDK CCEH consortium, this laboratory could be available to all of the institutions in the consortium, based on availability.
Please contact Diane Krause at Diane.Krause@yale.edu.
Expert Consultations: Cell Preparation and Analysis Core
Expert consultations are available from Dr. Jun Lu, Dr. Manoj Pillai, and Dr. Diane Krause