Equine Stem Cells. Sources, Processing, Expansion, Storage and Shipping

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1 Equine Stem Cells Sources, Processing, Expansion, Storage and Shipping Sean Owens, DVM, Diplomate ACVP Medical Director, UC Davis Veterinary Regenerative Medicine Laboratory

2 What is a Stem Cell and What Constitutes Regenerative Medicine?! A stem cell is a progenitor (mother) cell that can differentiate into multiple cell types! Regenerative Medicine is the process of using those cells to create living functional tissues to repair or replace damaged tissues! The promise of stimulating previously irreparable tissues and organs to heal themselves through biological medicine

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4 Equine Adult Stem Cell Sources! Umbilical Cord " Blood " Tissue Fat Bone Marrow

5 Umbilical Cord Sources! We have collected umbilical cord blood and tissue from over 90 horses and demonstrated that it is safe for both mare and foal.! Two comparative populations in harvested versus non harvested foals we found no difference in: " Time to stand and nurse " Time for mare to pass placenta " 24 hr foal blood counts Bartholomew et, al. EVJ, 2009

6 Umbilical Cord Blood Collection

7 Umbilical Cord Tissue Collection

8 Umbilical Cord Sources! Cord Blood: " A high volume of blood readily, ethically and easily collected " No age-related declines in MSC number or MSC differentiation potential " Probable higher proliferative potential and increased flexibility for differentiation " Lower numbers of MSCs than bone marrow, cord tissue and fat overall! Cord Tissue: " High numbers of highly proliferative MSCs " Noninvasive collection

9 Methods for Bone Marrow Collection! Bone marrow can be acquired from the sternum of adult horses.! In horses 5 years of age and under, bone marrow can be easily obtained from the hip.

10 Collection Technique - Sternum

11 Collection Technique - TC

12 Equine MSC Growth vs Location of Aspirate s l l e C f o # Tx Aspirate Location St p = (not significantly different) n = 4 horses

13 Equine MSC Growth - First vs Second Aspirate s l l e C f o # * * = p <0.05 n = 4 horses

14 Bone Marrow MSC Numbers! 0.02% of nucleated cells are stem cells! 1 in 4,000 to 10,000 cells! 2-3 week expansion: ~ million cells Vidal et.al., Vet Surg, 35, 2006

15 Bone Marrow Derived Stem Cells! Potency and number declines with age.! Maximum number and potency is most likely achieved in collections from horses under 18 months of age.! Stem cells from juvenile bone marrow collection can easily be processed, expanded and stored for future use.

16 Isolation of Stem Cells from Adipose Tissue (ASCs)

17 ASC Numbers! 4-40% ASCs in SVF! ,000 SVF cells/ml of fat! 40,000-2 million ASCs in 10 ml! 2-3 week expansion: ~ comparable to MSC from bone marrow Vidal et.al., Vet Surg, 36, 2007

18 Fat Derived Stem Cells! Stem cells can easily be expanded and stored for use as with bone marrow if adequate amounts of fat are obtained.! Collection of an adequate amount of fat can be problematic in young and fit racehorses.! Culture and expansion is required to achieve large numbers of MSCs for therapeutic use.

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20 Stem Cell Processing & Storage Overview Sample Shipping Pre-Expansion Processing Expansion/Culture Cryopreservation/Freezing Quality Assurance and Quality Control Regulatory Considerations

21 What s In The Sample? Bone Marrow & UCB Mononuclear Cells Stem Cells Lymphocytes Monocytes Neutrophils Red Blood Cells Non-Cellular Fraction Hematopoietic Stem Cells Mesenchymal Stem Cells

22 Stem Cell Transport Primary Considerations Shipping Temperature Optimal pre-shipping and shipping storage temperature prior to processing determined to be 4 C Shipping Container Selection Monitoring During Transport Data Log Shipment Tracking

23 Stem Cell Transport to the Processing Laboratory Shipping Container Temperature Probe Shipper The overall goal is to be able to ship your stem cells in a rapid, reliable manner in a safe and temperature controlled environment.

24 Stem Cell Processing! Primary Consideration: " Method of Processing! None, Ficoll, or Automated Ficoll Automated

25 Stem Cell Processing Other factors of importance Adequacy of sample (volume, viability) Nucleated cell counts Lack of equine stem cell markers Reporting of results Who, What and When Bacterial culture testing prior to release

26 Post-Processing Expansion/Culture! Plate and Incubate! Select for MSC population! Feed every 3 days until colony formation! Passage cells! Continue expansion till adequate cell numbers obtained Mixed Cells Confluent MSCs

27 Cord Tissue Processing! Disruption of cord tissue for cell isolation was labor intensive, tedious, time consuming and involved repetitive motion " Limited our ability to process high numbers of cord tissues for MSC isolation! Cord tissue-derived MSCs! Embryologically young, highly proliferative, high number of MSCs compared to cord blood, broader differentiation potential than fat or BM (?)

28 Polytron tissue homogenizer

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30 The Final Steps

31 Cord Tissue processing! Polytron disruption of cord tissue is fast, safe, relatively inexpensive and results in successful isolation of high numbers of MSCs! Modifications in tissue disruption and enzymatic digestion have decreased labor, time and cost associated with isolation of MSCs from cord tissue! Cord tissue processing changes will facilitate research! Isolation of MSCs from Cord tissue will be offered this season by the UC Davis Regenerative Medicine Laboratory

32 Storage! Important points " Method of cryopreservation

33 Cryopreservation Systems Controlled Rate Freezer Or Liquid Nitrogen Dewar System All in one system

34 Storage & Shipping Important points Method of Storage (-80 freezer vs. Liquid nitrogen) Evaluation of post-thaw viability Number of cells in the dose being shipped to you Cost to thaw and prepare for shipping Shipping vial selection It Does Matter Method of return shipping (Tracking & Accountability)

35 Optimum Storage Conditions for Long Term Cell Viability! Under Liquid Nitrogen (-196 C ) " No significant loss of clonogenic capacity after storage for years.! -80 C Mechanical Freezer MNC Viability 6 month 12 month 24 month 31 month 80% % Galmes et al Recommendation: No longer than 6 months storage at -80 C

36 Quality Control and Quality Assurance! Good Manufacturing Practice (GMP)! Establishment of scientifically sound and appropriate " Standards " Specifications " Test procedures " Identification of samples in relation to donor and recipient! Adequate provisions for monitoring the reliability, accuracy, precision and performance of the above

37 Regulatory Oversight in the Future As veterinary stem cell treatment becomes more popular and is used with greater frequency in the future, it is possible that the FDA and the USDA may take steps to regulate the industry and the practice. If this happens, companion animal stem cell treatment and its associated products could be regulated as biologics, requiring registration with the USDA, or drugs requiring FDA approval.

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