Re-aggregation Medium for Cryopreserved SC-islets
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Solution Overview
Problem
Current methods for cryopreserving and thawing human pluripotent stem cell-derived islets (SC-islets) result in significant cell death, unfavorable cluster morphologies, and poor islet health and function, limiting their utility in diabetes cell replacement therapy.
Innovation Solution
A re-aggregation medium comprising a basal medium with specific components such as MCDB131, Glutamax, penicillin-streptomycin, bovine serum albumin, glucose, ZnSO4, DNase I, and BI-6C9 is used for thawing and culturing SC-islets, which improves cell survival, growth, and differentiation, leading to high-recovery yields and maintained Beta cell identity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional cryopreservation and thawing methods are used for SC-islets, then the process is simple and quick, but cell death is significant and islet health and function are poor
Solution Approach 1:
The patent applies parameter changes by systematically optimizing the composition of the thawing and culture medium. Specific parameters including glucose concentration (0.115 g/L), ZnSO4 concentration (523 μL), DNase I concentration (1 mg/mL with 1:1000 dilution), and BI-6C9 concentration (10 μM with 1:4000 dilution) were adjusted to achieve high cell recovery yields and maintained beta cell identity while resolving the contradiction between simplicity and effectiveness.
2Productivity
If standard culture media are used for thawed SC-islets, then the media composition is simple, but cell recovery yield is low and clustering morphology is unfavorable
Solution Approach 1:
The patent employs composite materials by creating a complex re-aggregation medium that combines multiple components with specific functions: basal medium MCDB 131 provides foundational nutrients, Glutamax supplies amino acids, bovine serum albumin provides protein protection, glucose offers energy substrate, ZnSO4 provides trace elements, DNase I degrades DNA to improve morphology, and BI-6C9 inhibits apoptosis. This composite formulation achieves high cell recovery yields and favorable clustering morphology.
Solution Approach 2:
The patent uses intermediaries to resolve the contradiction between media simplicity and cell recovery effectiveness. DNase I acts as an intermediary enzyme that degrades DNA to improve clustering morphology, while BI-6C9 serves as an intermediary apoptosis inhibitor that protects cells during the critical thawing period. These intermediary substances enable high productivity without requiring complex procedural changes.
3Stability of the object's composition
If cryopreserved SC-islets are thawed using conventional methods, then the procedure is fast, but Beta cell identity is lost and insulin expression decreases
Solution Approach 1:
The patent applies preliminary action by incorporating apoptosis inhibitor BI-6C9 and DNase I into the re-aggregation medium before cell thawing. These pre-prepared protective agents are already present in the medium at optimal concentrations, enabling immediate protection of Beta cell identity and insulin expression during the thawing process, thus maintaining stability without extending the required time.
Solution Approach 2:
The patent ensures continuity of useful action by using a re-aggregation medium that continuously supports Beta cell identity and insulin expression throughout the entire thawing and initial culture period. The medium components work synergistically and continuously: glucose provides sustained energy, albumin provides continuous protein protection, and the apoptosis inhibitor maintains continuous cell protection, eliminating the need for multiple medium changes or extended culture times.
Data Source
AI summary
Among the various aspects of the present disclosure is the provision of a media for cryopreserved cells and methods of making and using same. An aspect of the present disclosure provides for a cell media formulation comprising (e.g., for day 0) one or more components selected from: MCDB 131; Glutamax; P/S; BSA; Glucose; ZnSO4; an enzyme for digesting DNA (e.g., DNASE1); and/or an apoptosis inhibitor (e.g., BI-6C9).