Bottlenecks in Clinical Source Material Acquisition. Aby J. Mathew, PhD May 5, 2009 ISCT Annual Meeting San Diego, CA

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1 Bottlenecks in Clinical Source Material Acquisition Aby J. Mathew, PhD May 5, 2009 ISCT Annual Meeting San Diego, CA

2 Biopreservation What s the issue? Biopreservation considerations towards Source Material Acquisition Unmet needs Scientific support for improved biopreservation 2

3 HISTORICAL ASSUMPTIONS Assumes that the cell/tissue is a passive participant Assumes that the effects of the sub lethal stresses are not additive or reversible Assumes cell in = cell out Assumes that Perceived Viability immediately post preservation = True Viability long term 3

4 BIOPRESERVATION YIELD THE PROBLEM! Cascading yield effects common to nearly all biopreservation applications Outcomes heavily affected by lack of in process efficiencies and optimization Does the beginning affect the end? Where does my cell therapy end up? 4

5 Low yield (viability and function) of source material and manufactured products often from current preservation technologies and processes Limited shelf life of source material and finished products Quality Concerns Need to reduce or eliminate toxic components Need for defined media with few/no animal components Varied/misleading results from assays and time of testing Impact of preservation process on outcomes Biopreservation media solutions are excipients/reagents in Cell Therapy Has your commercial media or self formulated cocktail been fully qualified in your process? Does your preservation solution actually work? Is your cell therapy product really as effective (including cost effective) as you want? Risk Management Suboptimal biopreservation affects each phase of development Source Material Acquisition Transportation Logistics R&D Cell Banking Therapeutic Delivery Clinical Efficacy Cost Effectiveness POINTS TO CONSIDER 5

6 BIOPRESERVATION: UNMET NEEDS & OPTIMIZATION GOALS Extending shelf life, improving transportation logistics Increasing post preservation recoverable yield Increasing post preservation functional yield Improving biopreservation media quality Improving efficiency in media prep Source material 6

7 RECENT STUDY IN CYTOTHERAPY

8 Cellular responses to low temperature what happens? Metabolic activity decreases Ionic and osmotic balance is disrupted reduced membrane pump activity Free radicals accumulate Molecular stress responses are initiated Initiation of apoptotic cell death signaling pathways Secondary necrotic injury (necroptosis) CELLULAR RESPONSE TO COLD Multiple points for preservation stress (hypothermia, ischemia, postpreservation) Cellular priorities are survival, recovery, then functional performance 8

9 Adjustment of Na, K, Ca, Mg ratios to restrict passive ion movement FORMULATING A SOLUTION Osmotic Support impermeant anion to replace Cl in the extracellular space and balance fixed intracellular ions Chelators buffer calcium, iron dysregulation ph Buffering Capacity specific to low temperature (15ºC to 2ºC) Free Radical Scavengers Energy substrates but still serum free! Inhibition of preservation induced Apoptosis and Necrosis Intracellular like vs. Extracellular like 9

10 MOLECULAR ORIGIN OF CELL DEATH Mathew et al.,

11 IMPROVED HYPOTHERMIC STORAGE Media HTS FRS 11

12 EXPANDED PRESERVATION WINDOW 24 Hour 48 Hour 12

13 Biopreservation solutions are considered Excipients Not a Drug Not a Medical Device Is it cgmp Manufactured? Are there FDA Master Files available for cross reference? Does it have Fully Defined Components and Finished Product? Protein Free Serum Free USP/Multicompendial/Highest quality components QUALITY AND REGULATORY Elevate the bar for Biopreservation solutions 13

14 VISUAL EVIDENCE 5 Days Storage of Human Fibroblasts at 2 8ºC and 1 Day Recovery at 37ºC NHDF Cells Stored in HypoThermosol NHDF Cells Stored in Viaspan NHDF Cells Stored in Celsior Green = Actin Cytoskeleton Red = Mitochondria Activation Blue = Nuclear Stain 14

15 WHAT DO YOUR CELLS LOOK LIKE? 24 hr storage at 2-8ºC in intracellular-like HypoThermosol 24 hr storage at 2-8ºC in traditional media Human Keratinocytes stained for mitochondrial activity (red) and cytoskeletal integrity (green) Snyder et al.,

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