CN105018425B

CN105018425B - A kind of peripheral blood lymphocytes culture medium of animal origin-free ingredient

Info

Publication number: CN105018425B
Application number: CN201510399372.0A
Authority: CN
Inventors: 邱壮伟; 丘力功; 倪世明
Original assignee: GUANGZHOU BAIYUNSHAN BAIDI BIOTECHNOLOGY CO Ltd
Current assignee: GUANGZHOU BAIYUNSHAN BAIDI BIOTECHNOLOGY CO Ltd
Priority date: 2015-07-09
Filing date: 2015-07-09
Publication date: 2018-09-28
Anticipated expiration: 2035-07-09
Also published as: CN105018425A

Abstract

The invention discloses a kind of peripheral blood lymphocytes culture medium of animal origin-free ingredient, mainly composition includes：Basal medium, L-type phytohemagglutinin, compound amino acid, sodium selenite, ironic citrate, rh-insulin and buffer system.Serum of the peripheral blood lymphocytes culture medium of the animal origin-free ingredient of the present invention without containing animal or people, avoid safety problem caused by using animal blood serum possible, production cost can be reduced simultaneously, enhance the quality stability between product different batches, cell culture effect is promoted, safe and reliable reagent is provided for clinical examination.

Description

A kind of peripheral blood lymphocytes culture medium of animal origin-free ingredient

Technical field

The present invention relates to a kind of peripheral blood lymphocytes culture mediums of animal origin-free ingredient.

Background technology

With cytogenetic development, Chromosome In Peripheral Blood Lymphocytes karyotyping has become the routine of pre-natal diagnosis Detection project.And the split coil method cell for karyotyping more than number is obtained, the in vitro culture proliferation of lymphocyte is base Plinth.

Cell injuring model system simulate in vitro cell in vivo ambient growth and establish, and from animal or Person is that the serum of the mankind is widely used in maintenance and the proliferation of cell.In serum containing cell survival and growth necessary at Point：Growth factor, cell factor, adhesion factor, albumen, vitamin, trace element and hormone etc., serum is also used as buffering Agent, chelating agent.In addition, the physics that the albumin in serum, myosin and other albumen can influence cell culture system is special Property, such as pH, shearing force, viscosity, osmotic pressure and gas transport etc..

However, using serum, there is also many problems.Expensive, serum price rises steadily, and expense accounts for cultivating Base cost half；Serology Quality is irregular, and there are unstable quality, can lead to the difference of production and product quality；In addition, There are mycoplasmas and other viral heterologous to pollute in serum, the safety issue of biological product is caused, although at can be through inactivation Reason, still, such processing can influence the promoting growth of cell ability of serum.

In order to solve the problems, such as serum in extensive utilization, enterprise be increasingly turned to serum-free, without animal derived ingredient very The exploitation of culture medium is determined to chemical composition.

Invention content

The purpose of the present invention is to provide a kind of peripheral blood lymphocytes culture mediums of animal origin-free ingredient

The technical solution used in the present invention is：

A kind of peripheral blood lymphocytes culture medium of animal origin-free ingredient, mainly composition include：Basal medium, L Type phytohemagglutinin, compound amino acid, sodium selenite, ironic citrate, rh-insulin, buffer system.

Preferably, the basal medium is RPMI1640 culture mediums.

Preferably, the compound amino acid includes：Pidolidone 300mg/L, L- glycine 100mg/L, L- asparagus fern Propylhomoserin 400mg/L, 360 mg/L of Serine, 80 mg/L of L-threonine, 80 mg/L of L-Leu, 200 mg/ of L-PROLINE L。

Preferably, the buffer system is NaHCO₃。

Preferably, the content of each component is in the culture medium：Basal medium 10-10.5 g/L, L-type plant blood Ball agglutinin 18-22 mg/L, -22 ml/L of amino acid compound 18, sodium selenite 50-70 n mol/L, ironic citrate 0.5- 1.5 mg/L, rh-insulin 8-12 mg/L, buffer system 1.8-2.2 g/L.

The beneficial effects of the invention are as follows：

Serum of the peripheral blood lymphocytes culture medium of the animal origin-free ingredient of the present invention without containing animal or people, avoids Using animal blood serum may caused by safety problem, while production cost can be reduced, enhanced between product different batches Quality stability, promoted cell culture effect, safe and reliable reagent is provided for clinical examination.

Specific implementation mode

With reference to embodiment, the present invention is further illustrated, and however, it is not limited to this.

Embodiment 1

1, the preparation of 1L span amino acid mother liquors：The sweet ammonia of Pidolidone 300 mg, L- is separately added into 500 mL waters for injection 100 mg of acid, 400 mg of L-Aspartic acid, 360 mg of Serine, 80 mg of L-threonine, 80 mg, L- dried meat ammonia of L-Leu 200 mg of acid are settled to 1.0L after stirring and dissolving.4 DEG C of storages are spare after packing.The culture medium of embodiment 1-3 uses this ammonia Base acid mother liquor.

2, the preparation method of the peripheral blood lymphocytes culture medium of 1L animal origin-frees ingredient is：In 800mL waters for injection 2.0 g of sodium bicarbonate, span amino acid mother liquor 20 mL, PHA- is added in 1640 culture medium dry powders of middle addition RPMI, 10.4 g after dissolving 20 mg/L of L, 10 mg of 60 nmol/L of sodium selenite, 1 mg of ironic citrate and rh-insulin, stirring make fully to dissolve, and use 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxides adjust pH value between 7.0-7.4, and 0.22 um membrane filtration degermings divide Dress, -20 DEG C of storages are spare.

Embodiment 2

The preparation method of the peripheral blood lymphocytes culture medium of 1L animal origin-free ingredients is：In 800mL waters for injection 1640 culture medium dry powders of RPMI, 10 g is added, 2.2 g of sodium bicarbonate, 18 mL, PHA-L 22 of span amino acid mother liquor are added after dissolving Mg/L, 12 mg of 50 nmol/L of sodium selenite, 0.5 mg of ironic citrate and rh-insulin, stirring make fully to dissolve, and use 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxides adjust pH value between 7.0-7.4, and 0.22um membrane filtration degermings dispense ,- 20 DEG C of storages are spare.

Embodiment 3

The preparation method of the peripheral blood lymphocytes culture medium of 1L animal origin-free ingredients is：In 800mL waters for injection 1640 culture medium dry powders of RPMI, 10.5 g is added, 1.8 g of sodium bicarbonate, span amino acid mother liquor 22 mL, PHA-L are added after dissolving 18 mg/L, 8 mg of 70 nmol/L of sodium selenite, 1.5 mg of ironic citrate and rh-insulin, stirring make fully to dissolve, and use 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxides adjust pH value between 7.0-7.4, and 0.22um membrane filtration degermings dispense ,- 20 DEG C of storages are spare.

4 lymphocyte transformation rate of embodiment measures

Lymphocyte culture is carried out using the embodiment 1-3 culture mediums prepared and the comparison culture medium containing calf serum.

Comparing culture medium prescription is：1640 basal medium 9.36g/L of RPMI, calf serum 10% (W/V), PHA-L 20mg/L, heparin 0.1% (W/V).

Experimental procedure：

Serum-free cell culture medium and each one bottle of culture medium of comparison are taken, aseptically uses 2ml syringes that 20 drops are added dropwise Anticoagulation gently shakes up, and is put into 37 ° of incubators and places 72 hours, every 12 hours jogs 1 time.Harvest uses 1ml in first 2 hours Syringe is added dropwise to colchicin, and final concentration of 0.02 ug/ml shakes up in postposition incubator and continues to cultivate.With pipettor by nothing Serum cell culture medium moves into 10ml centrifuge tubes, 1000 revs/min, centrifuges 5 minutes, abandons supernatant, cell is dripped after fixation Suspension is on slide, push jack.It is dyed with Wright's staining liquid after piece is dry, cell is in sky blue.1000 monokaryons are counted under oil mirror Cell, including conversion and unconverted cell.

Lymphocyte transformation rate calculation formula：It drenches and converts lymphocyte number/1000*100% in the cell of rate of rotation=1000.

Measurement result is shown in Table 1：

Table 1

The experimental results showed that, culture medium of the present invention equally can in the case where not adding the serum of animal or people above The culture effect for promoting cell, safe and reliable reagent is provided for clinical examination.

Above example is only to introduce the preferred case of the present invention, to those skilled in the art, without departing substantially from this Any obvious changes and improvements carried out in the range of spirit are regarded as the part of the present invention.

Claims

1. a kind of peripheral blood lymphocytes culture medium of animal origin-free ingredient, consisting of：Basal medium 10-10.5 g/ L, L-type phytohemagglutinin 18-22 mg/L, -22 ml/L of amino acid compound 18, sodium selenite 50-70 n mol/L, lemon Lemon acid iron 0.5-1.5 mg/L, rh-insulin 8-12 mg/L, buffer system 1.8-2.2 g/L；It is characterized in that, described Basal medium is RPMI1640 culture mediums；The compound amino acid includes：Pidolidone 300mg/L, L- glycine 100mg/ L, L-Aspartic acid 400mg/L, 360 mg/L of Serine, 80 mg/L of L-threonine, 80 mg/L, L- dried meat ammonia of L-Leu 200 mg/L of acid.

2. the peripheral blood lymphocytes culture medium of animal origin-free ingredient according to claim 1, which is characterized in that described Buffer system is NaHCO₃。