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Three-dimensional culture method of palatal organs in vitro

A three-dimensional culture and organ technology, applied in the field of cell and molecular biology, to achieve the effect of low cost and simple operation

Active Publication Date: 2018-04-17
DALIAN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no matter which culture method is used, the longest culture time of palatal plates in vitro is only 80 hours

Method used

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  • Three-dimensional culture method of palatal organs in vitro
  • Three-dimensional culture method of palatal organs in vitro
  • Three-dimensional culture method of palatal organs in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Matrigel three-dimensional culture of palatal plate tissue in vitro

[0029] The middle one-third of the facial tissues (all the tissues from the infraorbital rim to the corner of the mouth, including the palate, the lower part of the nasal septum and surrounding tissues, referred to as palatal organs) were separated from the normal mouse embryonic palate development period (embryonic day 5). Mix with Matrigel Matrigel diluted 1-fold or 2-fold in serum-free DMEM, 10 samples per group, and place in an incubator to solidify into a gel. After 1 hour, DMEM medium containing 10% fetal bovine serum was added for incubation. Unembedded mouse palate tissue was used as the experimental control group (10 samples). All groups were cultured in vitro for 6 days, and fresh culture medium was replaced every two days. After 6 days, the palatal plate tissue of each group was observed, the number of samples with normal palatal plate tissue structure, bilateral palatal plate ...

Embodiment 2

[0035] Example 2: Collagen hydrogel three-dimensional culture of palatal plate tissue in vitro

[0036] Separation of tissues from the middle third of the palate during embryonic development of spontaneous cleft palate mice (day 6 of embryonic stage) (all tissues from the infraorbital rim to the corner of the mouth, including the palate, the lower part of the nasal septum and surrounding tissues, referred to as palatal organs) , and then mixed with mouse type II collagen (molecular weight 500kDa) solutions with a concentration of 5 mg / ml and 10 mg / ml respectively, 10 samples in each group, and put them in an incubator to solidify for 40 minutes to form a cylindrical hydrogel with a diameter of 2 cm and a thickness of 1 cm. Glue, and added DMEM medium containing 10% fetal bovine serum for culture. The unembedded palate tissue of mice with spontaneous cleft palate was used as the experimental control group (10 samples). All groups were cultured in vitro for 6 days, and fresh cu...

Embodiment 3

[0041] Example 3: In vitro three-dimensional culture of palatal plate tissue on oxidized hyaluronic acid / collagen composite hydrogel scaffold

[0042] Bovine sodium hyaluronate (molecular weight 1100 kDa) was prepared into a 10 mg / ml aqueous solution, and 20 mM sodium periodate solution was added according to the volume ratio of sodium hyaluronate / sodium periodate at 4:1, and stirred at 25°C in the dark. React for 5 hours, then dialyze, and finally freeze-dry to obtain oxidized hyaluronic acid.

[0043] Prepare 0.9% (w / v) collagen solution, and mix the collagen solution, DMEM medium and NaOH-NaHCO 3 -Hepes buffer is mixed according to the ratio of 7:2:1 (v / v), and then 0.6% (w / v) oxidized hyaluronic acid aqueous solution is mixed according to 10:1 and 30:1 (v / v) (solution: oxidized transparent Aqueous acid solution) was mixed, stirred slowly, and the mixture was placed in a refrigerator at 4 °C for 24 h.

[0044] Separation of the mid-third of the facial tissues of the mouse...

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Abstract

The invention discloses a three-dimensional culture method for palatal organs in vitro. The whole tissue from the inferior orbital edge to the corner of the mouth is taken from the embryonic palate in the early stage of development or in the developmental stage, embedded in a hydrogel support, and culture solution is added for three-dimensional culture. It can provide a three-dimensional growth environment similar to the body, avoid the use of expensive materials and processes, and has the advantages of simple operation and low cost. In addition, the biological characteristics of the palatal plate tissue can be adjusted by changing the composition of the hydrogel scaffold, which is suitable for different stages of embryonic development The tissue culture of normal and abnormal palate plate can be used for the study of the formation mechanism of cleft palate and the screening of drugs that promote the formation of palate plate.

Description

technical field [0001] The invention relates to the fields of cell and molecular biology, in particular to a three-dimensional palatal organ culture method in vitro that can realize long-term high activity of palatal plate tissue. Background technique [0002] Cleft lip and palate are relatively common developmental deformities in newborns, and animals with cleft palate are difficult to survive after birth. In order to study the development process of the palate plate and the mechanism of the fusion of the palate plate, it is very necessary to culture the palate plate tissue in vitro. At present, there are only two methods of in vitro culture of palatal plate tissue: static culture and dynamic culture: the static culture method is to place the palate plate on a porous membrane, and add medium for static culture; the dynamic culture method is to suspend in the state of culture fluid flow nourish. However, no matter which culture method is used, the longest culture time of pa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
Inventor 肖晶孙广炜刘洋徐小溪李楠刘波丛蔚马小军
Owner DALIAN MEDICAL UNIVERSITY
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