Method of obtaining deposited lymphokine-activated killer cells

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to methods of obtaining deposited lymphokine-activated killer cells (LAKC) for treatment of oncologic diseases. Claimed is method, according to which mononuclear lymphocytes (MNL) are isolated from peripheral blood of malignant effusion by centrifugation on one-step gradient of ficoll with density 1.06-1.08 g/cm3, LAKC are generated and concentrated by successive precipitation of MNL by centrifugation, re-slurring it in RPMI 1640 or DMEM medium with addition of 2-10% AB human serum, interleikin-2 in concentration 0.5-1.0 mlnIU/ml and incubation in CO2 incubator for 48-72 hours, after that suspension of obtained LAKC is deposited in culture medium in amount 2 mln LAKC per 200 ml of medium on sterilised and washed with culture medium porous titanium carriers with 55-60% porosity.

EFFECT: invention allows to increase efficiency of obtained medication for local and local-regional immunotherapy of oncologic patients.

1 ex, 8 dwg, 2 tbl

 

The invention relates to medicine, namely to methods immunorehabilitation of cancer.

Known methods of immunotherapy allow you to get from light fractions of mononuclear leukocytes (ISL) of the patient's blood, a significant number of so-called lymphokine-activated killer cells (LAK). LAC-immunotherapy widens the range of possibilities of anticancer therapy. In addition, it has advantages over traditional methods: non-toxicity, good tolerability of treatment, the possibility of use in conjunction with other traditional therapeutic methods; in cases of drug resistance, stimulation of local antitumor cellular immunity leads to lysis of the tumor, but also improves the quality and duration of life of patients. However, as research has shown, bearsdley LACQUER in a systemic injection of activated lymphocytes predominantly accumulate in the lung and liver and is defined in the area of the tumor nodules in these bodies is not more than 1 day. Therefore, to increase the effectiveness of immunotherapy used repeated administration of large doses of VARNISH (up to several billion per course). High-dose IL-2/LAK-therapy is accompanied by severe side effects (fever, chills, artrell what I myalgia, nausea, vomiting, diarrhea, disorders of vascular permeability and impaired respiratory function). In recent years, developing methods of local and Loco-regional adaptive immunotherapy, allowing you to create an effective concentration of the VARNISH in the field of tumor process and minimize systemic side effects. Because not all cases there is a possibility of targeted delivery LACQUER to tumor formations, promising is the development of methods allowing to form in various tissues depot activated cells. Produced in the process of life deposited LACQUER cytokines may exert long-term stimulatory effect on antitumor immunity. In addition, recently there was information about the ability of the VARNISH is introduced into tumor sites, to provide not only a direct cytotoxic effect on tumor cells, but also by secreted cytokines to recruit in the region of introduction of VARNISH other effectors of antitumor immunity. It is important to note that the VARNISH can lyse tumor cells not only by direct contact, but also by distant actions of cytokines, inducing the release cytopathogenic factors (e.g., nitrous oxide) from macrophages. The structural basis for the creation of such a depot can serve as porous titanium the new plates, which is a product of the us-technology (from the English. scaffold - scaffolding), which are currently widely used in surgical and orthopaedic practice.

Closest to the claimed method is a method of obtaining cell transplant for the treatment and prevention of cancer, infectious diseases and immunodeficiency, characterized by the fact that blood or a suspension of bone marrow is centrifuged in a single-stage gradient ficoll, conduct the cultivation of MNCs in medium RPMI 1640 or DMEM with 5% human serum of the patient or AB serum of the donor, the cells are divided into monocytes/macrophages, is attached to the substrate, and lymphocytes, are not attached to the substrate, MNCs are placed in culture medium for 2-4 h and then poured neprecejusies lymphocytes to obtain immature DC, the remaining adherent cells to monocytes/macrophages add granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), then to stimulate DC maturation use of Pro-inflammatory factors IL-1β, TNF-αIL-6 (10 ng/ml), obtained in in vitro conditions, immature dendritic cells (CD83low, CD86low, CD80low) incubated with tumor lysate, or viral or bacterial antigens and factors in the induction of DC maturation within 1 day and get Mature pulsed with antigens DK (CB83 high, CD86high, CD80highfor LAC-cells to lymphocytes add IL-2 at a concentration of 1000 IU/ml (see patent RU 2309753, CL AC 35/14, publ. 10.11.2007). The disadvantages of this method are the need to use multiple injections of large quantities of lymphocytes, as well as the need for local or locoregional introduction. This significantly complicates the application of this method in cancer patients with tumors of different localization.

The invention aims to remedy these disadvantages.

The technical result consists in increasing the efficiency of resources for local anaesthesia and locoregional anaesthesia immunotherapy of cancer patients. The problem is solved and the technical result is achieved by the fact that according to the method of obtaining deposited lymphokine-activated killer cells (LAK) was isolated mononuclear lymphocytes (ISL) from peripheral blood or malignant effusion by centrifugation on a single-stage gradient ficoll density of 1.06-1.08 g/cm3generate and concentrate LACQUER by successive deposition of the ISL by centrifugation, resuspendable it in medium RPMI 1640 or DMEM with the addition of 2-10% AB human serum, Il-2 at a concentration of 0.5-1.0 million IU/ml and incubation in CO2-incubator for 48-72 h, and then the depot is niroot suspension cells obtained VARNISH in the culture medium at the rate of 2 million LAC 200 ml of medium in sterilized and washed with culture medium porous titanium media with porosity 55-60%.

Figure 1-4 shows the histogram reflecting immunophenotype LACQUER,

figure 1 - distribution for CD16+;

figure 2 - distribution for CD56+,

figure 3 - distribution for CD25+;

figure 4 - distribution of CD38+.

When taken designation: Gm - geometric mean deviation of the signal, CV - coefficient of variation; in brackets - registering channels, after the brackets is the average channel, in parentheses, the percentage of cells expressing this antigen, M1 - level values different from the control.

Figure 5-8 shows the cytotoxic activity of VARNISH deposited in scaffold, when the magnification of 400 times.

figure 5 - intact cells K-562;

figure 6 - incubation of intact VARNISH with cells K-562;

7 - intact cells K-562 and empty us;

on Fig - incubation of VARNISH deposited in the us), with cells L-562.

The allocation of the ISL from peripheral blood can be done in various ways, for example by centrifugation in a single-stage gradient ficoll. To prevent blood clotting stabilize commonly used for these purposes, preservatives, such as, for example, heparin, citrate and EDTA.

Accom is emy method allows to obtain a sufficient number of lymphocytes from 100 ml of peripheral blood or from one dose of lakomski donor, that greatly simplifies fence mononuclear leukocytes holding hardware separation of blood.

The cultivation of the ISL is in RPMI-1640 or DMEM with 2-10% human serum of the patient (or AB serum donor) in disposable plastic bottles with a volume of 250 ml of the firm Costar or similar bottles from other companies. To obtain lymphokine-activated killer cells ISL cultured with interleukin-2 in a concentration of 0.5-1.0 IU/ml

As scaffolds use of porous titanium plate porosity 55-60%, which has the shape of parallelepipeds (15×3×3 mm). Sterilized and washed with culture medium scaffold strongly shaken to free the pores from the remnants of the environment and on the surface of the faces put 2 million LACQUER in 200 µl of culture medium.

An example embodiment of the invention.

1. The allocation of the ISL from peripheral blood.

ISL extracted from stabilized by heparin (25 units/ml) peripheral blood for single-stage gradient ficoll ("Pharmacia", density 1.077 g/cm3) by centrifugation at 400 g for 30 minutes. Lymphoid cells, interphase formed ring, collected by pipette and washed three times in medium 199. After each washing 10 times the volume of medium, the cells are precipitated by centrifugation at 200 g.

2. Generation of LACQUER.

ISL count, precipitated by centrifugation and resus jirout in medium RPMI 1640 with additives (HEPES, L-glutamine, gentamicin) in a concentration of 1 million/ml, with the addition of 5% AB human serum, Il-2 at a concentration of 0.5-1.0 million IU/ml and incubated in CO2-incubator for 48-72 hours As shown in figure 1, the population of activated ISL predominantly by natural killer cells (CD16+CD56+and activated forms of lymphocytes (CD25+, CD38+).

3. Pick up VARNISH in porous titanium scaffold.

Sterilized and washed with culture medium scaffold was intensively shaken to free the pores from the remnants of the environment and deposited on the surface of the faces 2 million LACQUER in 200 µl of culture medium.

4. Microscopy.

Intact VARNISH and LACQUER deposited titanium scaffold and placed in 24-hole tablets, viewed and photographed on invertoscope Axiovert (Zess, Germany). As shown in figure 2 microscopic examination, LACQUER, placed in the us, causing more pronounced lysis of cell line K 562, than intact VARNISH. Whilst there was no evidence of dispersion VARNISH from scaffold; the hole was observed only isolated lymphoid elements.

5. Evaluation of antitumor activity of intact VARNISH and LACQUER deposited in the porous titanium scaffold, on the line of tumor cells To 562.

In the wells of a 24-hole tablet ("Costar, USA) were made by 2 million LACQUER or the us with LACQUER and ito line To 562. In the control series in the wells of the microplate was added only NAIL Polish, us, loaded LACQUER or cells To 562. Cells were incubated for 48 h at 37°C and 4.5% CO2. Then selected part of the environment for subsequent determination of cytokines.

The impact of scaffolds on the anticancer properties of the VARNISH were evaluated using the MTT colorimetric test. Index cytotoxic activity (ICA) VARNISH was calculated on the basis of optical density in the control and experimental series. The optical density was measured on multiskan MS (Labsystem, Finland). The cytotoxic effect LACQUER in scaffold was the result of a non-contact exposure effectors to target cells. Submissions suggest that the VARNISH in the us longer retain their cytotoxic activity and after 10 days of cultivation are significantly higher biocidal activity compared to intalniri LAC (average 22%) (table 1).

6. Determination of cytokine production intact VARNISH and LACQUER deposited in the porous titanium scaffold.

The concentration of cytokines in the culture medium was determined using a set to study the concentration of cytokines person FlowCytomix human Th1/Th2 11plex BMS810 FF (Bender MedSystems) method of flow cytofluorometry on the FACSCalibur instrument Systems.

Table 2 presents data on production of cytokines LACQUER. The mouth is owino, the impact of metal scaffold no effect on the secretion of cytokines by activated lymphocytes. The VARNISH is placed in scaffold continue to function adequately as the concentration of cytokines in the incubation medium, practically identical to that of the intact VARNISH. However, the paint is deposited in scaffold produce anti-inflammatory cytokine IL-10 in smaller quantities.

Table 1
The change in the index of cytotoxic activity (ICA, %) intact and placed in the us VARNISH when tested on the cell line To 562
EffectorsTiming of cultivation VARNISH (d)
2510
Intact VARNISH88±374±462±5
LACQUER in us89±186±284±2*
* significant (p<0.05) difference from the intact VARNISH

Table 2
Cytokine production LACQUER (PCG/ml)
Cells-producersINFγIL-6IL-4IL-5IL-1βTNFαTNFβIL-10IL-12p70IL-2
Intact VARNISH6601±726,11751±1576,600±19540±70,21508±90,564±3,8887±79,816±3,01625±260,0
LAC SCAF.5879±649,71878±1691,000±19500±65,01630±of 97.843±2,6243±21,920±2,51504±240,6
Empty the SCAF.+LACQUER4190±are 460.91045±938,30 0±18485±63,1987±59,220±1,2791±71,213±10,21026±164,2
To 5620±11,028±5,200±184±0,511±0,70±6,10±9,00±18,96±16,2
* significant (p<0.05) difference from intedned LACQUER

The method of obtaining deposited lymphokine-activated killer cells (LAK), which emit mononuclear lymphocytes (ISL) from peripheral blood or malignant effusion by centrifugation on a single-stage gradient ficoll density of 1.06-1.08 g/cm3generate and concentrate LACQUER by successive deposition of the ISL by centrifugation, resuspendable it in medium RPMI 1640 or DMEM with the addition of 2-10% AB human serum, interleukin-2 in a concentration of 0.5-1.0 mlnl/ml and incubation in CO2incubator for 48-72 h, and then deposited by a suspension of the obtained cell LA is in the culture medium at the rate of 2 million Polish on 200 ml of medium in sterilized and washed with culture medium porous titanium media with porosity 55-60%.



 

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