Optimised and defined method for isolation and preservation of precursor cells from human umbilical cord

一种分离细胞、人脐带的技术，应用在细胞解离方法、生物化学设备和方法、胚胎细胞等方向，达到效力可靠的效果

Inactive Publication Date: 2010-10-20
MEDINFAR PROD FARM
View PDF8 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the solutions described in the prior art to date lack assurance, the present invention is expected to alleviate the need for a method of the character described above

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Optimised and defined method for isolation and preservation of precursor cells from human umbilical cord
  • Optimised and defined method for isolation and preservation of precursor cells from human umbilical cord
  • Optimised and defined method for isolation and preservation of precursor cells from human umbilical cord

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1: Optimizing the type and amount of mechanical operation and strip size

[0123] Optimized for type of mechanical manipulation and size of initial tissue fragments, maintaining tissue mass (g), bottom surface area of ​​digestion flask (cm 2 ), digestion volume (ml), and total bottle volume (ml) at a constant ratio of about 1:2:2:37, considering that a 1 cm human umbilical cord strip weighs about 1 g. After removal of the amnion, several types of cord segments were tested: low (5 cm segments); medium (2.5 cm segments); high (0.3 cm segments); and minced tissue. All segmentation was performed with the aid of a scalpel and the samples were treated with essentially the same conditions. It was concluded that the best yield in terms of total cells / cord mass / time at the end of P0 was obtained when 2.5 cm strips were used. Table 1 summarizes the quantitative results obtained.

[0124] Table 1: Segmentation Optimization: Cell Yield

[0125] Low (5cm)

[...

Embodiment 2

[0127] Example 2: Advantages of blood clots in umbilical cord vessels (1 vein and 2 arteries) change

[0128] Lysis of red blood cells is known to be toxic, reducing cell viability in vitro. Therefore, cell yields were compared when digestions were performed with and without blood clots. For the latter experiment, blood clots were removed with the aid of a scalpel. It was concluded that blood clots had a negative effect on yield in terms of total cells / cord mass / time at the end of P0. Table 2 summarizes the quantitative results obtained.

[0129] Table 2: Blood clot: Effect on cell yield

[0130] Have

[0131] Note: +++ = excellent, ++ = very good, + = good, - = fair, -- = poor, 0 = unsuccessful

Embodiment 3

[0132] Example 3: Properties of enzymes in digestive solutions, enzyme action alone or in combination, and Optimization of Enzyme Concentration

[0133] In order to maximize the yield in terms of the number of cells isolated from the initial tissue with the desired characteristics, two initial approaches were taken with respect to enzymatic digestion: in the presence of culture medium, without digestion, and thus in the absence of enzymes, the cells adhered directly to the culture flask, And the tissue was dissociated with a single enzyme: 0.075% (w / v) collagenase II or 2.0% (w / v) pronase.

[0134] Since collagenase II alone was the most effective method, this enzyme was combined with other enzymes, especially with 0.125% (w / v) trypsin (with or without 0.260 mM EDTA), with only 0.5% (w / v) hyaluronidase in combination, and in combination with a combination of 0.5% (w / v) hyaluronidase and 2.0% (w / v) pronase.

[0135] For this test, an optimized strip size of 2.5 cm was used...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The present invention refers to an optimized and defined method for isolation and preservation of precursor cells from human umbilical cord. Besides being reproducible and 100% reliable, in terms of the number of samples processed, the method results in a high and defined number of precursor cells, being the majority obtained after a single adhesion and expansion / multiplication phase ex vivo (thus granting cell phenotype), in a shorter time frame than what was previously described in the state-of-the-art. With this method, it is possible to obtain, in 9 days, after direct freezing of a cell fraction, and after one expansion / multiplication phase ex vivo (end of PO) of the majority of the cells, about 8, 6, 6 (+ / -0, 1) x l05 cells / gram of processed umbilical cord. In turn, the characteristics of the cells allow, for example, after 35 days, obtaining an average of 7.7xlO15 cells, with precursor phenotype, from 100% of processed umbilical cord samples. The method, because it is simple, robust and 100% reliable, can be performed under good manufacturing practices (GMP) in laboratories dedicated to cell therapy in humans. Furthermore, the method has applications in the pharmaceutical, cosmetic and biotechnology areas.

Description

technical field [0001] The present invention relates to an optimized and established method of isolating and preserving precursor cells from human umbilical cord. [0002] "Prerequisite cells" in the context of the present invention refer to a class of cells capable of attaching and expanding / proliferating on surfaces and in defined growth media, where the majority of cells in culture express the cell surface markers CD44, CD73, CD90 and CD105 , and most of the cells showed only residual expression of cell surface markers CD14, CD31, CD34 and CD45, and most of them were able to undergo up to 18 expansion / proliferation phases, maintaining a constant doubling factor of about 1.7 / 24 hours , constant fibroblast-like morphology, and the ability to partially or terminally differentiate into specialized cells such as osteoblasts, chondrocytes, adipocytes, cardiomyocytes, and glial / neural cells. [0003] The object of the present invention adds to the prior art a robust method that a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/073C12N5/0775
CPCC12N5/0668C12N2509/00C12N5/0605A61P43/00
Inventor R·I·冈查斯索雷斯M·C·巴普提斯塔克尔豪J·M·希尔瓦桑图斯J·P·玛丁斯V·A·巴斯图P·埃斯提利塔蒙提罗达克鲁兹H·J·索雷斯达克鲁兹
Owner MEDINFAR PROD FARM
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap