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Method for isolating stromal vascular fraction

A technique of stromal vascular components and homogenization, applied in the field of molecular biology, can solve problems such as safety concerns, increased contamination risks, and difficult quality control

Inactive Publication Date: 2016-04-27
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of enzymes such as collagenase may raise safety concerns because collagenase is usually produced from the Gram-positive bacterium Clostridium histolyticum
Using an enzyme also makes quality control difficult due to its shelf life and batch-to-batch variability in its activity
Additionally, collagenase digestion requires long processing times (approximately 1 hour) and often results in inconsistencies in yield expressed in cell numbers
Considering the use of collagenase of bacterial origin, the method also requires extensive washing, which leads to an increase in the overall time and cost involved in the separation or recovery process
In addition, methods known in the art also require extensive manual handling by the technician, which increases the risk of contamination

Method used

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  • Method for isolating stromal vascular fraction
  • Method for isolating stromal vascular fraction
  • Method for isolating stromal vascular fraction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 - Preparation of post-harvest samples

[0059] method

[0060] Adipose tissue obtained as a lipoaspirate or surgically resected is obtained prior to performing any of the methods as described herein. To ensure optimal cell viability, it is recommended to isolate the stromal vascular fraction immediately on the day the sample is obtained. Optimally, however, the methods as described herein can be performed within 24 hours of obtaining the sample. If the methods of the present disclosure cannot be performed immediately upon harvesting the sample or within 72 hours of obtaining the sample, the sample is stored at 4°C. In the case of enzymatic digestion, allow the samples to reach room temperature before proceeding to the isolation procedure.

[0061] When obtaining a sample as a lipoaspirate, to remove debris, the adipose tissue is washed with an equal volume of buffer, such as phosphate-buffered saline (PBS) or Hanks' balanced salt solution (HBSS). For examp...

Embodiment 2

[0064] Example 2 - Traditional Enzymatic Methods

[0065] Known enzymatic methods for recovering stromal vascular components have been described previously (WO2011 / 032025; Sugii et al., 2010 and Sugii et al., 2011). Briefly, the known / traditional enzymatic method involves the following steps:

[0066] i) Add an equal volume of collagenase solution (ml / gram or cc fat pad) to a sterile vial and warm to 37°C. Typically, the collagenase solution consists of 2.5 μg / ml type I collagenase, 1% (wt / vol) bovine serum albumin (BSA), 50 μg / ml D-glucose and 200 nM adenosine in buffer;

[0067] ii) placing the washed adipose tissue in a collagenase solution;

[0068] iii) finely mincing the tissue with sterilized scissors;

[0069] iv) Shake the vial in a 37°C water bath shaker (about 100 rpm) for 30-60 min. Check the digest every 10 minutes, and stop the reaction when the reaction is complete;

[0070] v) Transfer and filter the digested solution through a 250 μm nylon filter. Then f...

Embodiment 3

[0078] Example 3 - Ultrasonic Treatment Method (Water Bath Ultrasonic Apparatus)

[0079] When using ultrasonic treatment method, especially water bath sonicator, the detailed embodiment of the step of the method of the present disclosure is as follows:

[0080] i) placing the washed adipose tissue in an appropriately sized tube;

[0081] ii) Fill with cooled water to the level shown to avoid overheating of the water bath sonicator;

[0082] iii) Sonicate the adipose tissue sample for 5-10 min at the lowest level setting (eg, 40 kHz, using Branson's model 3510). It will be apparent to those skilled in the art that different sonicators will give very different results. Therefore, optimization of sonication time and intensity will be required;

[0083] iv) The samples were centrifuged at 400 g for 5 min to pellet the dissociated stromal vascular fraction cells. Transfer the supernatant to a new tube and use it in step vi) below if necessary;

[0084] v) Resuspend the pellet...

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Abstract

Disclosed is a method for recovering stromal vascular fraction from a sample. The method comprises providing a mechanical force capable of breaking the sample into a single cell suspension whilst maintaining intact stromal vascular fraction cells cellular structures, wherein the mechanical force is mincing and / or homogenization. Also disclosed are methods of using the stromal vascular fraction.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of priority from Singapore Patent Application No. 2013053095 filed on 10 July 2013, the contents of which are hereby incorporated by reference in their entirety for all purposes. technical field [0003] The present invention relates to the field of molecular biology. In particular, the invention relates to devices for use in cell separation. Background technique [0004] Stem cells are an ideal resource for developing regenerative therapies. In general, stem cells exist in limited numbers in the human body, and protocols to fully optimize their purification and use efficiency are still under development. Adipose tissue has been increasingly recognized as a promising source of stem cells potentially beneficial for regenerative medicine. In addition to mature adipocytes, adipose tissue contains relatively abundant populations of progenitor and mesenchymal stem cells (MSCs) within t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K35/36A61K35/12A61P3/10A61P37/00A61P29/00A61P17/02A61P43/00
CPCA61K35/12A61K35/36C12N2527/00C12N5/0667A61P17/02A61P29/00A61P37/00A61P43/00A61P3/10A61K35/35C12N5/0653
Inventor 杉井重纪王伟杰
Owner AGENCY FOR SCI TECH & RES
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