CN105018425A

CN105018425A - Animal derived component-free peripheral blood lymphocyte medium

Info

Publication number: CN105018425A
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: 2015-11-04
Anticipated expiration: 2035-07-09
Also published as: CN105018425B

Abstract

The invention discloses an animal derived component-free (ADCF) peripheral blood lymphocyte medium. The medium contains the following major components: a basic medium, an L-type phytohemagglutinin, compound amino acid, sodium selenite, ferric citrate, recombinant human insulin and a buffer system. The ADCF peripheral blood lymphocyte medium contains no animal or human serum. Thus, the safety problem which might be caused by use of animal serum is avoided. Meanwhile, product production cost can be reduced, mass stability of different batches of products is enhanced, and cell culture effect is raised. A safe and reliable reagent is provided for clinical examination.

Description

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

Technical field

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

Background technology

Along with cytogenetic development, Chromosome In Peripheral Blood Lymphocytes karyotyping has become the conventional sense project of antenatal diagnosis.And the split coil method cell of many Gong the karyotypings of number will be obtained, lymphocytic vitro culture propagation is basis.

Cell injuring model system is analog cell ambient growth and setting up in vivo in vitro, and the serum deriving from animal or the mankind is widely used in maintenance and the propagation of cell.Containing cell survival and the necessary composition of growth in serum: somatomedin, cytokine, adhesion factor, albumen, VITAMIN, trace element and hormone etc., serum can also as buffer reagent, sequestrant.In addition, the albumin in serum, Pp63 glycophosphoproteins and other albumen can affect the physical property of cell culture system, as pH, shearing force, viscosity, osmotic pressure and gas transport etc.

But, use serum also to there is many problems.Expensive, serum price rises steadily, and expense accounts for culture medium cost half; Serology Quality is uneven, there is quality unstable, can cause the difference of production and quality product; In addition, there is mycoplasma and other viral heterologous in serum and pollute, cause the safety issue of biological product, although can through inactivation treatment, such process can affect the promoting growth of cell ability of serum.

In order to solve serum in the problem extensively used, enterprise has turned to serum-free, gradually without animal derived the composition even exploitation of chemical composition determination substratum.

Summary of the invention

The object of the present invention is to provide a kind of peripheral blood lymphocytes culture medium of animal origin-free composition

The technical solution used in the present invention is:

A peripheral blood lymphocytes culture medium for animal origin-free composition, it mainly forms and comprises: basic medium, L-type phytoh(a)emagglutinin, aminoacids complex, Sodium Selenite, ironic citrate, recombinant human insulin, buffer system.

As preferably, described basic medium is RPMI1640 substratum.

As preferably, described aminoacids complex comprises: Pidolidone 300mg/L, L-glycine 100mg/L, L-Aspartic acid 400mg/L, Serine 360 mg/L, L-threonine 80 mg/L, L-Leu 80 mg/L, L-PROLINE 200 mg/L.

As preferably, described buffer system is NaHCO ₃.

As preferably, in described substratum, the content of each component is: basic medium 10-10.5 g/L, L-type phytoh(a)emagglutinin 18-22 mg/L, amino acid compound 18-22 ml/L, Sodium Selenite 50-70 n mol/L, ironic citrate 0.5-1.5 mg/L, recombinant human insulin 8-12 mg/L, buffer system 1.8-2.2 g/L.

The invention has the beneficial effects as follows:

The serum of peripheral blood lymphocytes culture medium not containing animal or human of animal origin-free composition of the present invention, avoid the safety problem using animal serum to cause, products production cost can be reduced simultaneously, strengthen the quality stability between product different batches, promote cell cultures effect, for Clinical Laboratory provides safe and reliable reagent.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.

embodiment 1

1, the preparation of 1L span amino acid mother liquor: add Pidolidone 300 mg, L-glycine 100 mg, L-Aspartic acid 400 mg, Serine 360 mg, L-threonine 80 mg, L-Leu 80 mg, L-PROLINE 200 mg respectively in 500 mL waters for injection, be settled to 1.0L after stirring and dissolving.After packing, 4 DEG C of storages are for subsequent use.The substratum of embodiment 1-3 all uses this span amino acid mother liquor.

2, the compound method of the peripheral blood lymphocytes culture medium of 1L animal origin-free composition is: in 800mL water for injection, add RPMI 1640 culture medium dry powder 10.4 g, sodium bicarbonate 2.0 g, span amino acid mother liquor 20 mL, PHA-L 20 mg/L, Sodium Selenite 60 nmol/L, ironic citrate 1 mg and recombinant human insulin 10 mg is added after dissolving, stirring makes abundant dissolving, with between 0.1 mol/L hydrochloric acid and 0.1 mol/L sodium hydroxide adjust ph to 7.0-7.4,0.22 um membrane filtration is degerming, packing ,-20 DEG C of storages are for subsequent use.

embodiment 2

The compound method of the peripheral blood lymphocytes culture medium of 1L animal origin-free composition is: in 800mL water for injection, add RPMI 1640 culture medium dry powder 10 g, sodium bicarbonate 2.2 g, span amino acid mother liquor 18 mL, PHA-L 22 mg/L, Sodium Selenite 50 nmol/L, ironic citrate 0.5 mg and recombinant human insulin 12 mg is added after dissolving, stirring makes abundant dissolving, with between 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide adjust ph to 7.0-7.4,0.22um membrane filtration is degerming, packing ,-20 DEG C of storages are for subsequent use.

embodiment 3

The compound method of the peripheral blood lymphocytes culture medium of 1L animal origin-free composition is: in 800mL water for injection, add RPMI 1640 culture medium dry powder 10.5 g, sodium bicarbonate 1.8 g, span amino acid mother liquor 22 mL, PHA-L 18 mg/L, Sodium Selenite 70 nmol/L, ironic citrate 1.5 mg and recombinant human insulin 8 mg is added after dissolving, stirring makes abundant dissolving, with between 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide adjust ph to 7.0-7.4,0.22um membrane filtration is degerming, packing ,-20 DEG C of storages are for subsequent use.

embodiment 4 lymphocyte transformation rate measures

The substratum adopting embodiment 1-3 to prepare and the contrast substratum containing calf serum carry out lymphocyte cultivation.

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

Experimental procedure:

Get serum-free cell culture medium and contrast each one bottle of substratum, aseptically drip 20 anticoagulations with 2ml syringe, shake up gently, put into 37 ° of incubators placements 72 hours, every 12 hours jogs 1 time.Gather in the crops and be added dropwise to colchicine with 1ml syringe in first 2 hours, final concentration is 0.02 ug/ml, shakes up in rearmounted incubator and continues to cultivate.With pipettor, serum-free cell culture medium is moved into 10ml centrifuge tube, 1000 revs/min, centrifugal 5 minutes, abandon supernatant liquor, after fixing, drip cell suspension on slide, push jack.With the dyeing of Wright's staining liquid after sheet is dry, cell is sky blue.Under oil mirror, counting 1000 monocytes, comprise and transforming and unconverted cell.

Lymphocyte transformation rate calculation formula: drench rate of rotation=1000 transit cell lymphocyte number/1000*100%.

Measurement result is in table 1:

Table 1

Above experimental result shows, substratum of the present invention, when not adding the serum of animal or human, can promote the culture effect of cell equally, for Clinical Laboratory provides safe and reliable reagent.

Above embodiment is only introduces preferred case of the present invention, to those skilled in the art, not deviating from any apparent changes and improvements of carrying out in the scope of spirit of the present invention, all should be regarded as a part of the present invention.

Claims

1. a peripheral blood lymphocytes culture medium for animal origin-free composition, it mainly forms and comprises: basic medium, L-type phytoh(a)emagglutinin, aminoacids complex, Sodium Selenite, ironic citrate, recombinant human insulin, buffer system.

2. the peripheral blood lymphocytes culture medium of animal origin-free composition according to claim 1, is characterized in that, described basic medium is RPMI1640 substratum.

3. the peripheral blood lymphocytes culture medium of animal origin-free composition according to claim 1, it is characterized in that, described aminoacids complex comprises: Pidolidone 300mg/L, L-glycine 100mg/L, L-Aspartic acid 400mg/L, Serine 360 mg/L, L-threonine 80 mg/L, L-Leu 80 mg/L, L-PROLINE 200 mg/L.

4. the peripheral blood lymphocytes culture medium of animal origin-free composition according to claim 1, is characterized in that, described buffer system is NaHCO ₃.

5. the peripheral blood lymphocytes culture medium of the animal origin-free composition according to any one of claim 1-4, it is characterized in that, in described substratum, the content of each component is: basic medium 10-10.5 g/L, L-type phytoh(a)emagglutinin 18-22 mg/L, amino acid compound 18-22 ml/L, Sodium Selenite 50-70 n mol/L, ironic citrate 0.5-1.5 mg/L, recombinant human insulin 8-12 mg/L, buffer system 1.8-2.2 g/L.