Adipose Tissue Cryopreservation via DMSO and Trehalose

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Solution Overview

Problem

Current methods for cryopreserving human adipose tissue derivatives are inadequate in maintaining tissue vitality and structural properties over long periods, leading to inefficiencies in clinical procedures and waste of valuable biological material, as they fail to preserve the regenerative potential and integrity of the tissue.

Innovation Solution

A method involving a controlled freezing protocol using a mixture of dimethyl sulfoxide (DMSO) and trehalose, followed by a specific thawing protocol with successive washes of decreasing DMSO concentrations, which helps in maintaining the integrity and viability of adipose tissue samples.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of stationary object

If adipose tissue is cryopreserved using conventional methods, then the tissue can be stored for long periods, but the cell viability and structural integrity are compromised

Engineering Contradiction:
Improvestorage durationVSAvoidcell viability
Core Design Contradiction:
Duration of action of stationary objectVSReliability

Solution Approach 1:

The patent applies parameter changes by optimizing the freezing rate (controlled at 1-2°C per minute) and using a two-stage freezing process (initial freezing at -80°C followed by liquid nitrogen storage at -196°C). These controlled parameter changes prevent intracellular ice crystal formation while maintaining cell membrane integrity, thus preserving cell viability during long-term storage

Inventive Principle:
Principle #35Parameter changes

Solution Approach 2:

The patent uses cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO) and trehalose as intermediaries. These substances mediate between the freezing process and the biological tissue by preventing ice crystal formation, reducing osmotic stress, and maintaining cellular structure during cryopreservation, thereby preserving cell viability

Inventive Principle:
Principle #24Intermediary (Mediator)

2Duration of action of stationary object

If adipose tissue is cryopreserved using conventional methods, then the tissue can be stored for long periods, but the structural properties and regenerative potential are lost

Engineering Contradiction:
Improvestorage durationVSAvoidstructural integrity
Core Design Contradiction:
Duration of action of stationary objectVSStability of the object's composition

Solution Approach 1:

The patent employs controlled parameter changes including a two-stage freezing protocol (initial slow freezing at 1-2°C per minute to -80°C, then transfer to liquid nitrogen at -196°C) and optimized thawing rates. These parameter controls prevent structural damage by avoiding rapid ice crystal formation that would disrupt the extracellular matrix and vascular-stromal niche architecture

Inventive Principle:
Principle #35Parameter changes

Solution Approach 2:

Cryoprotective agents (DMSO and trehalose) serve as intermediaries that protect structural integrity by forming protective complexes around cellular structures, preventing ice crystal formation, and maintaining the architecture of the vascular-stromal niche during freezing and storage

Inventive Principle:
Principle #24Intermediary (Mediator)

3Ease of operation

If a simple thawing protocol is used, then the process is quick and easy, but the tissue vitality and regenerative properties are compromised

Engineering Contradiction:
Improvethawing simplicityVSAvoidtissue vitality
Core Design Contradiction:
Ease of operationVSReliability

Solution Approach 1:

The patent applies preliminary action by pre-warming the thawing buffer to 37°C before use and preparing all necessary materials in advance. The thawing protocol itself is simplified to a single step (immersion in pre-warmed buffer) rather than requiring gradual warming or multiple steps, thus maintaining both simplicity and effectiveness

Inventive Principle:
Principle #10Preliminary action

Solution Approach 2:

A specialized thawing buffer containing protective agents serves as an intermediary during the thawing process. This buffer mediates the transition from frozen to thawed state by preventing thermal shock, maintaining osmotic balance, and protecting cellular structures, thereby preserving tissue vitality through a simple one-step process

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

The method achieves improved cell viability and structural integrity of adipose tissue after long-term storage, reducing the need for repeated liposuction procedures and enhancing the flexibility and effectiveness of regenerative medicine treatments.

Implementation Method 1

A method involving a controlled freezing protocol using a mixture of dimethyl sulfoxide (DMSO) and trehalose

Methodology Applied
Scientific EffectCryoprotection: Freezing

Implementation Method 2

a controlled freezing protocol using a mixture of dimethyl sulfoxide (DMSO) and trehalose

Methodology Applied
Scientific EffectFreezing: Freezing

Implementation Method 3

followed by a specific thawing protocol with successive washes of decreasing DMSO concentrations

Methodology Applied
Scientific EffectThawing: Melting

Data Source

PatentEP3994984B1A method for the cryopreservation of samples of adipose tissue
Publication Date: 2023.05.24 LIPOBANK SRL
  • EP3994984B1 patent drawingFigure 1~2
  • EP3994984B1 patent drawingFigure 3~4b
  • EP3994984B1 patent drawingFigure 5

AI summary

The present invention relates to a method for the cryopreservation of human adipose tissue samples, comprising a tissue freezing step and a thawing step after a storage period, wherein the particular thawing conditions allow a high rate of cell viability to be maintained, while preserving the integrity and functionality of the freshly harvested tissue.