Sand-Mediated Ice Seeding for Low-Toxicity hiPSC Cryopreservation

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Solution Overview

Problem

Current methods for cryopreserving human induced pluripotent stem cells (hiPSCs) are hindered by high toxicity from cryoprotectants like DMSO and serum, which can cause differentiation and introduce xenogeneic factors, compromising cell quality and viability.

Innovation Solution

A method utilizing sand-mediated ice seeding at temperatures above −10°C to control ice nucleation, minimizing cryoprotectant use and ensuring high cell survival and pluripotency, using a sand-PDMS film in cryovials for controlled ice nucleation during cryopreservation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional cryopreservation methods using high concentration of DMSO and serum are used, then cell protection during freezing is improved, but cell toxicity and differentiation risk increase

Engineering Contradiction:

Improvecell protection during freezingVSAvoidcell toxicity and differentiation risk

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and removes the harmful components (DMSO and serum) from the cryopreservation medium, replacing them with a serum-free, DMSO-free formulation that uses alternative protective mechanisms (ice nucleation control and osmoprotectants) to achieve cell protection without toxicity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention changes the chemical composition parameters of the cryopreservation medium by eliminating DMSO and serum, and instead uses controlled ice nucleation temperature and alternative osmoprotectants to achieve protection, fundamentally altering the mechanism from chemical protection to physical control

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If slow-freezing method is used for hiPSC cryopreservation, then ease of operation is improved, but cell survival rate is limited to around 50%

Engineering Contradiction:

Improveconvenience of cryopreservationVSAvoidcell survival rate

Core Design Contradiction:

Ease of operationVSReliability

Solution Approach 1:

The invention applies preliminary action by pre-cooling the cryovial with sand-mediated ice nucleation before actual freezing begins, controlling the ice formation process in advance to prevent intracellular ice and optimize water efflux, thereby improving survival while maintaining ease of operation

Inventive Principle:

Principle #10Preliminary action

3Reliability

If vitrification method is used to achieve high cell survival, then cell protection is improved, but device complexity and protocol difficulty increase

Engineering Contradiction:

Improvecell protectionVSAvoidspecialized protocols and devices required

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention uses simple, inexpensive materials like sand and standard cryovials instead of specialized vitrification devices and complex equipment, achieving effective cryopreservation with disposable, low-cost components that simplify the overall system

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

4Object-affected harmful factors

If DMSO concentration is reduced to lower toxicity, then cell toxicity is decreased, but cryoprotection effectiveness is compromised

Engineering Contradiction:

Improvecell toxicityVSAvoidcryoprotection effectiveness

Core Design Contradiction:

Object-affected harmful factorsVSReliability

Solution Approach 1:

The invention introduces alternative mediators (sand-mediated ice nucleation and osmoprotectants like sugars) to replace DMSO's protective function, using these intermediary substances to control ice formation and protect cells without the toxic effects of DMSO

Inventive Principle:

Principle #24Intermediary (Mediator)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Achieves high viability (up to 90%) and pluripotency of cryopreserved hiPSCs with reduced DMSO concentration, maintaining differentiation capacity and reducing risks associated with conventional cryopreservation methods.

Implementation Method 1

sand-mediated ice seeding at temperatures above −10°C to control ice nucleation

Methodology Applied

Scientific EffectIce nucleation: Nucleation

Implementation Method 2

utilizing sand to seed ice for cryopreservation... sand-PDMS film in cryovials for controlled ice nucleation

Methodology Applied

Scientific EffectHeterogeneous nucleation: Nucleation

Data Source

PatentUS12382953B2System, device, and method for cell cryopreservation via sand-mediated ice seeding

Publication Date: 2025.08.12 UNIV OF MARYLAND

AI summary

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above −10° C., which enables cryopreservation of hiPSCs with no serum, minimized cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by the pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.