No-spin Cryopreservation Using Diluted DMSO

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Solution Overview

Problem

Current cryopreservation methods for living cells require post-thaw washing and centrifugation to remove cryoprotectants, which are time-consuming, costly, and prone to contamination, and limit throughput due to the need for skilled operators and sequential processing of cells.

Innovation Solution

A method for cryopreserving cells in a concentrated suspension with reduced cryoprotectant volume, allowing direct thawing and use without washing or centrifugation, by diluting the cryoprotectant to non-toxic levels during thawing, enabling direct plating or suspension without the need for post-thaw processing steps.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If standard cryopreservation method with high concentration cryoprotectant is used, then cell protection during freezing is improved, but post-thaw washing and centrifugation are required which increases process complexity and time consumption

Engineering Contradiction:

Improvecell protection during freezingVSAvoidpost-thaw processing complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The invention changes the concentration parameter of the cryoprotectant from high (10-12% DMSO) to low (0.5-2% DMSO), enabling the solution to protect cells during freezing while being non-toxic after thawing, thus eliminating the need for post-thaw washing and centrifugation steps

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention performs preliminary dilution of the cryoprotectant concentration before freezing, preparing a diluted freezing solution in advance that maintains protective function during freezing but becomes non-toxic after thawing, avoiding the need for post-thaw removal steps

Inventive Principle:

Principle #10Preliminary action

2Reliability

If standard cryopreservation method is used, then cell protection during freezing is improved, but throughput is limited due to sequential processing requirements

Engineering Contradiction:

Improvecell protection during freezingVSAvoidthroughput

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

By changing the cryoprotectant concentration parameter to a low level that is non-toxic after thawing, multiple vials can be processed simultaneously without requiring sequential washing and centrifugation, thereby increasing throughput while maintaining cell protection

Inventive Principle:

Principle #35Parameter changes

3Reliability

If high concentration cryoprotectant is used, then freezing protection is improved, but cell toxicity increases requiring removal steps

Engineering Contradiction:

Improvefreezing protectionVSAvoidcryoprotectant toxicity

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The invention changes the concentration parameter of the cryoprotectant from high (10-12%) to low (0.5-2%), creating a diluted freezing solution that provides adequate freezing protection while being non-toxic to cells after thawing, eliminating the need for cryoprotectant removal steps

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention uses a partially sufficient concentration of cryoprotectant (diluted to 0.5-2%) that provides adequate freezing protection without excessive toxicity, balancing protective function with cell safety after thawing

Inventive Principle:

Principle #16Partial or excessive action

4Quantity of substance

If post-thaw washing and centrifugation are performed, then cryoprotectant removal is improved, but handling risks and contamination probability increase

Engineering Contradiction:

Improvecryoprotectant removalVSAvoidcontamination risk

Core Design Contradiction:

Quantity of substanceVSReliability

Solution Approach 1:

By changing the cryoprotectant concentration to a low level (0.5-2%) that is non-toxic after thawing, the invention eliminates the need for post-thaw washing and centrifugation steps, thereby removing handling operations that could introduce contamination while maintaining adequate cryoprotectant removal through simple dilution

Inventive Principle:

Principle #35Parameter changes

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

This approach simplifies the thawing process, reduces handling risks, increases throughput, and maintains cell viability and functionality comparable to conventional methods, allowing for efficient and user-friendly use of cryopreserved cells in various applications.

Implementation Method 1

mixing the concentrated cell suspension with the freezing solution to yield a cryopreservation mixture

Methodology Applied

Scientific EffectDiffusion: Diffusion

Implementation Method 2

subjecting the cryopreservation mixture to cryopreservation

Methodology Applied

Scientific EffectFreezing: Freezing

Implementation Method 3

The cryoprotectants, including permeating and nonpermeating types, are essential components of freezing medium, since they prevent the formation of both intra- and extracellular ice crystals

Methodology Applied

Scientific EffectCryoprotection:

Implementation Method 4

The concentrated composition of cryopreserved cells is thawed to yield a thawed composition of cells

Methodology Applied

Scientific EffectThawing: Melting

Data Source

PatentUS10292382B2No-spin cryopreservation technique and resulting products

Publication Date: 2019.05.21 BIOPREDIC INT

AI summary

Methods and processes for cryopreservation and direct cell thawing and seeding or suspension after cryopreservation, including methods that eliminate the necessity of post-thaw wash, spin, and frequent practice of performing a cell count. Cell compositions and no-spin cell products produced using the methods are also described.