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Construction method for in vitro skin test model containing melanocytes

A melanocyte and test model technology, applied in the field of tissue engineering biomaterials, can solve the problem that skin test models are not suitable for long-term efficacy evaluation of drugs and cosmetics, and achieve parallelism and stability, low cost, and culture stability. Good results

Active Publication Date: 2017-10-24
GUANGDONG BOXI BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for constructing an in vitro skin test model containing melanocytes, which solves the problem that the skin test model constructed by the existing method is not suitable for people's long-term efficacy of drugs and cosmetics. evaluation question

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  • Construction method for in vitro skin test model containing melanocytes
  • Construction method for in vitro skin test model containing melanocytes
  • Construction method for in vitro skin test model containing melanocytes

Examples

Experimental program
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Effect test

Embodiment 1

[0049] Example 1 Construction of an in vitro skin test model containing melanin

[0050] Medium a provided in this example: KC-Growth as the base solution (1L), adding 0.2 μg / mL of hydrocortisone, 1.8 μg / mL of insulin, bovine pituitary gland extract 100μg / mL; and the addition of factors that play an important role in melanin production by melanocytes (hereinafter referred to as melanocytes), such as basic fibroblast growth factor 0.05ng / mL; make basal medium a.

[0051] Medium b: take medium a as the base solution (1 L), and add 0.47 mmol / L of calcium chloride, which plays an important role in the differentiation of keratinocytes, to prepare the base medium b.

[0052] The construction of the skin test model of the present embodiment is carried out according to the following steps:

[0053] Step 1. Construction of the skin test model epidermis containing melanin

[0054] Poly-lysine at a concentration of 25-75 μg / mL was used to coat the bottom of the chamber. Experiments sho...

Embodiment 2

[0057] Example 2 Construction of an in vitro skin test model containing melanin

[0058] The culture media a and b of this embodiment are the same as those in Embodiment 1.

[0059] The construction of the skin test model of the present embodiment is carried out according to the following steps:

[0060] Step 1. Construction of the skin test model epidermis containing melanin

[0061] The bottom of the chamber was coated with polylysine at a concentration of 25-75 μg / mL, and the keratinocytes of the 6th or 7th passage cultured in vitro were inoculated into the chamber with a cell suspension made of medium a. The inoculation ratio of keratinocytes and melanocytes is 1:10~1:1, and 1:5 is selected in this embodiment. The chamber inoculated with keratinocytes was then incubated at 37°C in 5% CO 2 Pre-cultivate in the incubator for 0.5 hours, and then inoculate the third or fourth generation melanocytes into a cell suspension using medium a (total amount of suspension = inoculat...

Embodiment 3

[0066] Example 3 Construction of an in vitro skin test model containing melanin with different skin colors

[0067] Medium a of this example: KC-Growth was used as the base solution (1L), and hydrocortisone 0.8 μg / mL, insulin 1.2 μg / mL, and bovine pituitary extract 50 μg / mL were added. mL; and adding factors that play an important role in producing melanin by melanocytes (hereinafter referred to as melanocytes), such as basic fibroblast growth factor 0.15ng / mL; make basal medium a.

[0068]Medium b: take medium a as the base solution (1 L), and add 0.6 mmol / L of calcium chloride, which plays an important role in the differentiation of keratinocytes, to prepare the base medium b.

[0069] The construction of the skin test model of the present embodiment is carried out according to the following steps:

[0070] Step 1. Construction of the skin test model epidermis of different skin colors containing melanin

[0071] The bottom of the chamber was coated with polylysine at a con...

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Abstract

The invention discloses a construction method for an in vitro skin test model containing melanocytes. The construction method comprises the following steps: using a culture medium for human epidermal keratinocytes as a basic liquid, and respectively preparing a culture medium a and a culture medium b; respectively preparing the keratinocytes and the melanocytes into cell suspensions by adopting the culture medium a, respectively and uniformly inoculating into a Transwell chamber, then adding the culture medium a, carrying out immersed culture for 1-5 days in a 5% CO2 incubator at the temperature of 37 DEG C, and forming an epidermal layer structure containing the melanocytes; placing on the surface of a culture support in a culture dish, adding the culture medium b, carrying out gas-liquid level culture, putting into the 5% CO2 incubator at the temperature of 37 DEG C and culturing for 5-15 days, so that the skin test model containing the melanocytes is obtained. The construction method disclosed by the invention has the advantages that model stability is high, the melanocytes are more sensitive to external stimulation, the model can be accurately used for detection on safety and efficacy assessment of cosmetics, raw material cost is low, production cycle is short, stability is good, and the model can be produced in quantity.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering biomaterials, and in particular relates to a method for constructing an in vitro skin test model containing melanocytes. Background technique [0002] The skin is the largest human organ and can be roughly divided into three layers from outside to inside: epidermis, dermis and subcutaneous tissue. The epidermis includes the stratum corneum and the basal layer. The stratum corneum is the outermost layer of the skin, which can withstand certain physical, chemical and mechanical injuries and protect the internal tissues. The basal layer is the lowest layer of the epidermis and is composed of basal cells and melanocytes. Melanocytes play the role of defenders in skin cells. Melanocytes resist the harmful effects of ultraviolet rays and environmental pollution on the skin by producing melanin granules. [0003] Cosmetics, pharmaceuticals and other products must undergo a large number of te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0626C12N5/0629C12N5/0698C12N2500/84C12N2501/115C12N2500/14C12N2503/06C12N2502/00C12N2501/33
Inventor 车景敏卢永波
Owner GUANGDONG BOXI BIO TECH CO LTD
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