HK1160679A - Stimulated cell standards - Google Patents

Abstract

A method for producing a reference standard sample for stimulating cells, which serves as a positive and negative control for monitoring intracellular cytokine levels and cytokine release in the test sample. The method for producing the reference standard sample comprises:; Stimulate cells to produce cytokines in the presence of cytokine release inhibitors, fix stimulated cells with fixatives such as paraformaldehyde, wash excess fixatives, and freeze dry the fixed stimulated cells.A method for producing labeled reference standard samples for cell proliferation analysis is also provided, which uses tags such as dyes to label proliferating mammalian cells isolated from human or animal bodies. During cell proliferation, progeny cells (such as carboxyfluorescein succinimide ester) can be distinguished, stimulated, and proliferated. The proliferated cells are fixed by adding fixatives and stored in freeze-drying or ultra-low temperature.

Description

Standard sample for stimulating cells

Technical Field

The present invention relates to a standard control reference material for cytokine and cell proliferation assays. The invention also relates to a method for producing such a material.

Background

The detection of cytokine release by Enzyme Linked Immunospot (ELISPOT), and the quantification and identification of intracellular cytokines by flow cytometry have been widely used as indicators of cellular immune responses. In the so-called enzyme-linked immunospot (ELISPOT), cytokines released from fixed cells typically undergo an immune reaction, such as enzyme-linked immunosorbent assay (ELISA), which shows a colored "spot" on the assay plate, thereby providing information on the identity (e.g., type of immune protein) and the quantity (number or proportion of reactive cells) of the cytokine. In order to standardize the assays for monitoring intracellular cytokine levels and cytokine release in test samples, it is necessary to develop reliable reference standards to allow comparability between different laboratories or assays.

Stimulated (cytokine positive), fixed, cryopreserved cells for intracellular flow cytometry analysis (Becton Dickinson, Oxford, UK) are commercially available for intracellular flow cytometry analysis (Becton Dickinson, Oxford, UK), while freeze-dried unstimulated cells are available for surface staining or hematology analysis (Beckman Coulter UK Ltd, High wyconbe, UK).

Currently, cytokine release from cells can be monitored by Enzyme Linked Immunospot (ELISPOT) or other assays, but only on cryopreserved living cells from individual donors.

The standardized reference materials provided by the currently available methods have several disadvantages: in order to make standardized reference materials available to a large number of laboratories worldwide, and to make the reference materials available for many years, a large amount of stable material is required. However, live cells from multiple donors cannot be mixed together in large single batches, as this would lead to mixed lymphocyte reactions, with consequent cell death and over-expression of cytokines.

Cryopreserved cells also require specialized storage, such as shipping with liquid nitrogen or dry ice, which increases costs. The freezer may also be subject to power outages, the risk of thawing and secondary freezing during shipment, causing the material to deteriorate and thus render the reference standard useless. Furthermore, cryopreserved cells must be carefully thawed to ensure consistency of the reaction. However, it has proven difficult to achieve consistent results across different laboratories using this approach.

Measuring cell proliferation of mammalian cells is also an important technique, such as the analysis of the effect of exogenous agents on the ability or propensity to differentiate cells. For example, such assay techniques can detect the proliferation of labeled immune system cells in an immune response. Many current techniques employ detectable labels that are labeled somewhere on the cell to differentiate progeny cells.

One such technique uses the fluorescent dye hydroxyfluorescein diacetate succinimide ester (CFSE or CFDA, SE). This technique is described in The paper published by Lyons, B. (immunological. cell Biol 1999; 77 (6): 509: "509. The technical can used in both video and video, available information to 10 confidential derivatives to cooled by flow cell, cell free detailed generation number can be used in functional analysis"). Other techniques include incorporation of bromodeoxyuridine or tritiated thymidine into cells.

While these analytical methods are well known, it is difficult to provide reference standard samples that can be calibrated against each other and quality controlled when these analytical techniques are used in different analytical laboratories or in sequential long-term analyses (e.g., clinical longitudinal studies).

The lack of reference materials for cytokine and cell proliferation assays has hindered the development of reliable testing methodologies, particularly in terms of blocking the comparability between different laboratories and assays. The present invention aims to solve these problems.

Disclosure of Invention

Accordingly, in a first aspect, the present invention provides a method of preparing a cellular reference material comprising the steps of: (a) obtaining a cell sample isolated from a human or animal body, said cell sample comprising cells selected from the group consisting of: basophils, neutrophils, eosinophils, monocytes, lymphocytes (B-and T-lymphocytes), natural killer cells (all peripheral blood mononuclear cells, PBMCs), thrombocytes and cell lines; (b) stimulating the production of cytokines by the cells in the presence of an inhibitor of cytokine secretion; (c) fixing said stimulated cells with a fixative; (d) freeze-drying the above fixed stimulated cells.

Using cell lines, we intend to be able to provide adequate fresh medium and space for the proliferation of cell cultures, which were originally isolated from the human or animal body and which are capable of producing cytokines. These cells may either produce cytokines themselves or may be modified by exogenous genetic material encoding, resulting in the synthesis of one or more cytokines, such as, but not limited to, TNF- α, IFN- γ, IL-1 and IL-6. Foreign DNA may be introduced into recipient eukaryotic cells by, for example, microinjection, electroporation, the use of calcium phosphate or lipofectin, and the above-mentioned genetic material may be present along with chromosomal DNA integrated into the cells, or may remain as plasmids. For cells that are originally capable of producing cytokines, these cells may also upregulate cytokine production by introduction of promoter sequences. Cells can be stimulated if desired, for example by production of cytokines using mitogens, and stored according to the techniques described herein.

Transduction of cytokine genes into tumor cells is known to cause a strong host inflammatory response (see "The boosting effect of co-transduction with cytokine genes on cancer therapy employing modified systemic inflammatory cells", International patent public of oncology; OJIMA Toshiyasu et al: ISSN 1019-. In vivo, this would impair tumor growth. In vitro, intracellular cytokines may be preserved according to the techniques described herein.

These cell lines may include cell lines derived from: basophils, neutrophils, eosinophils, monocytes, lymphocytes (B-and T-lymphocytes), natural killer cells (all peripheral blood mononuclear cells, PBMC), thrombocytes, or cell lines such as HeLa, HL-60, A-549, Jurkat, LNCap and CAPAN-1 cells.

In a preferred embodiment, the present invention provides a method of preparing a cellular reference material. The method comprises the following steps: (a) obtaining a cell sample isolated from a human or animal body, said cell sample comprising cells selected from the group consisting of: basophils, neutrophils, eosinophils, monocytes, lymphocytes (B-and T-lymphocytes), natural killer cells (all peripheral blood mononuclear cells, PBMCs) and thrombocytes; (b) stimulating the production of cytokines by the cells in the presence of an inhibitor of cytokine secretion; (c) fixing the stimulated cells by adding a fixing agent; (d) freeze-drying to preserve the fixed stimulating cells.

In a second, independent aspect, the present invention also provides a method of preparing a cellular reference material, comprising the steps of: (i) isolating a mixed population of cells from a human or animal body, said mixed population of cells comprising a plurality of cell types selected from the group consisting of: peripheral blood mononuclear cells, thrombocytes; (ii) separating said mixed population of cells into fractions, each fraction containing a distinct population of cells; (iii) subjecting one or more of the above components to cellular stimulation in the presence of an inhibitor of cytokine secretion to produce cytokines; (iv) adding a fixative to fix the cells in each fraction; (v) combining the above components to make mixed differential stimulation cells; (vi) the mixed differential stimulating cells are preserved by freeze-drying or ultra-low temperature.

In this way, reference standard samples can be produced that mimic specific patterns of cytokine expression in different cell types. These cytokine-expressed standards aid in clinical and research settings by aiding in the diagnosis/prognosis of underlying disease and the analysis of material/experimental results in the laboratory/experimental setting.

In any aspect of the invention, the cell sample is preferably isolated from a mammalian body, preferably from a human body.

In any aspect of the present invention, the cells are preferably stimulated by addition of a mitogen. Preferably, the intracellular cytokine is specifically stained simultaneously, and the mitogen includes PMA (phorbol 12-myristate 13-acetate). Also preferably, the mitogen comprises ionomycin. More preferably, the mitogen comprises a mixture of PMA (phorbol 12-myristate 13-acetate) and ionomycin. Also preferred, and particularly for use in Enzyme Linked Immunospot (ELISPOT), are mixtures of mitogens including PHA (phytohemagglutinin), IL-2 (interleukin-2), and a co-stimulatory anti-human CD28 monoclonal antibody.

In addition, in any one aspect of the present invention, the above cytokine secretion inhibitor preferably includes brefeldin a.

In any aspect of the present invention, the fixing agent preferably contains paraformaldehyde. More preferably, the fixative comprises a mixture of paraformaldehyde and chromium chloride. Immobilization is useful for halting biological activity, including apoptosis, inside and outside the cell, which improves the biological and structural integrity of the cell during lyophilization.

In addition, in any aspect of the invention, the method further comprises a step of removing the remaining fixative after the fixation of the cells and before the preservation. This allows the cells to be immediately reconstituted for use without the need for an additional washing step to remove the fixative. One benefit of this step is to minimize the volume change of the cells. Excess fixative can cause non-specific staining and prevent cytokine release in enzyme linked immunospot assays (ELISPOT).

In addition, in any aspect of the invention, the method further comprises a step of exposing said immobilized cells to a hypertonic environment prior to preserving said cells. By this step, the cells are in a state of water deficit in the body, making them completely more elastic, and these aspects also allow the overall yield of reference material to be increased.

In addition, in any aspect of the invention, the method additionally comprises a step of adding a cryoprotectant prior to freeze drying said cells. The addition of cryoprotectants is beneficial for improving the integrity of the cell structure during the freeze-drying process.

Overall, the benefits achieved by the present invention are:

(i) although the reference cells thus produced are most preferably stable at +4 ℃, the cells are capable of being transported at ambient temperatures without degradation;

(ii) the cells can be easily reduced with distilled water;

(iii) these cells can play a role in flow cytometry analysis and enzyme-linked immunospot analysis (ELISPOT), which release cytokines to the surrounding environment, particularly when maintained at high temperatures, around 37 ℃.

Furthermore, the scope of the present invention also includes a method for preparing a cellular reference material, which consists of the following steps: obtaining a plurality of cell samples isolated from a plurality of individuals; performing the above described method on each cell sample; prior to preserving said cells, additionally comprising a step of combining fixed cells derived from said plurality of samples. This has the added benefit of allowing large batches of samples from mixed donors to be prepared and stored, avoiding any mixed lymphocyte reactions that would otherwise occur with live cells in a mixed pool according to the methods described herein. Thus, a large number of standardized reference materials can be generated, which has not been possible in the past.

Furthermore, the scope of the present invention also includes a method for preparing a cellular reference material comprising the steps of: obtaining a plurality of mixed cell populations isolated from a plurality of individuals; performing the method as described above, each mixed cell population being treated according to the second aspect of the invention, further comprising a step of combining fixed cells derived from a plurality of said mixed cell populations before preserving said cells. This has the added benefit of quantitatively and qualitatively mimicking the synthesis/production of mixed lymphocyte populations and discovering various disease states from such mixed lymphocyte populations. For example, in the case of Graves' disease, thyroid cells express cytokines, which normally are not normally found, such as TNF- α, IFN- γ, IL-I, and IL-6.

Furthermore, the scope of the present invention also includes a method for preparing a cellular reference material comprising the steps of: preparing non-stimulated fixed cells according to the method steps of any one of the preceding aspects, but omitting the stimulating step (b) or (iii); combining the non-stimulated fixed cells with stimulated fixed cells prepared according to the method steps of any one of the preceding aspects; the combined cells are preserved by freeze-drying or ultra-low temperature method. This has the added benefit of providing a mixed lymphocyte population that mimics the non-diseased state and is compared to the diseased state population, i.e., as a negative control.

In a further independent aspect, the present invention also provides a method of preparing a cellular reference material comprising the steps of (i) isolating from a human or animal body a mixed population of cells comprising a plurality of cell types selected from the group consisting of: peripheral blood mononuclear cells, thrombocytes; (ii) fractionating said mixed population of cells to produce a fraction, said fraction having a preponderance of a single cell type; (iii) labeling cells of the fraction; (iv) fixing the labeled cells in the above-mentioned fraction by adding a fixing agent; (v) the labeled cells were stored by freeze-drying.

For example, CD4⁺Cells can be isolated from PBMCs using immunomagnetic bead sorting, stained with FITC (fluorescein isothiocyanate) to label anti-CD4 antibody, fixed as described herein and stored by lyophilization. These cells can be used as reference standard samples, for example, to calibrate the fluorescence of a flow cytometry apparatus.

In another aspect, the present invention provides a method of preparing a cellular reference material comprising the steps of: (a) obtaining proliferation-competent mammalian cells isolated from a human or animal body; (b) labeling said cells to differentiate progeny cells in the process of cell proliferation; (c) stimulating the proliferation of the labeled cells; (d) allowing said cells to proliferate; (d) fixing the proliferated cells by adding a fixing agent; (e) the resulting cells are stored in a freeze-dried or ultra-low temperature.

Preferably, the cells are stored by freeze-drying. Although effective, cryopreserved cells require specialized storage, such as shipping using liquid nitrogen or dry ice, which increases costs. The freezer may also be subject to power outages, the risk of thawing and secondary freezing during shipment, causing the material to deteriorate and thus render the reference standard useless. Furthermore, cryopreserved cells must be carefully thawed to ensure consistency of the reaction. Immobilization is useful for halting biological activity, including apoptosis, inside and outside the cell, which improves the biological and structural integrity of the cell during lyophilization.

Preferably, the cell is isolated from a mammal, and more preferably, the cell is isolated from a human.

The cells referred to in any aspect of the invention include proliferation-competent mammalian cells, preferably stimulated by the addition of a mitogen. Preferably, the mitogen contains PMA (phorbol 12-myristate 13-acetate). It is also preferred that the mitogen comprises ionomycin. More preferably, the mitogen comprises a mixture of PMA (phorbol 12-myristate 13-acetate) and ionomycin. In an alternative embodiment, stimulation of proliferation may be performed by the addition of mitogenic monoclonal antibodies such as UCHT 1.

Furthermore, in any aspect of the invention, the cells comprise proliferation-competent mammalian cells, and preferably the fixative comprises paraformaldehyde. More preferably, the fixing agent is prepared by mixing paraformaldehyde and chromium chloride.

Furthermore, in any aspect of the invention, the cells comprise proliferation-competent mammalian cells, preferably after fixing the cells and before preserving the cells, the method comprising an additional step of removing the remaining fixing agent. This allows the cells to be immediately reconstituted for use without the need for an additional washing step to remove the fixative. One benefit of this step is to minimize the volume change of the cells. Excess fixative can cause non-specific staining of the cell suspension.

Furthermore, in any aspect of the invention, said cells comprise proliferation-competent mammalian cells, preferably the method further comprises a step of exposing said immobilized cells to a hypertonic environment prior to preserving said cells. By this step, the cells are in a state of water deficit in the body, making them completely more elastic, and the intensification of these aspects also increases the overall yield of reference material.

Furthermore, in any aspect of the invention where the cells comprise proliferation-competent mammalian cells, it is preferred that the method additionally comprises a step of adding a cryoprotectant to the cells prior to preservation of the cells. The addition of cryoprotectants is beneficial for improving the integrity of cell structures during freeze-drying or cryopreservation.

In addition, in any aspect of the present invention, the above cells include proliferation-competent mammalian cells, and it is preferable that the above cells are labeled with a fluorescent marker. More preferably, the fluorescent label contains CFSE (carboxyfluorescein succinimide ester).

Also fully described herein is a method of making a cellular reference material encompassed by the scope of the present invention.

Detailed Description

EXAMPLE 1 production of Standard samples of stimulated cells

In one embodiment of the invention, the "buffy coat" is obtained from a source such as a venous blood donor or a national blood collection facility. The buffy coat fraction of the blood is the fraction of the blood that is obtained by centrifugation and contains the majority of white blood cells and platelets. All of these buffy coats obtained were subjected to routine testing prior to use to ensure negativity for treponema pallidum hemagglutination test ("TPHA"), hepatitis B surface antigen detection ("HBsAg"), anti-human immunodeficiency virus 1 ("anti-HIV 1"), anti-human immunodeficiency virus 2 ("anti-HIV 2"), and anti-hepatitis C virus ("anti-HCV") prior to use.

Residual red blood cells present in the buffy coat can be removed by any standard method using a commercially available lymphocyte density gradient formulation, such as a lymphocyte separating agent, followed by separation/collection of peripheral blood mononuclear cells ("PBMCs") and then a second application of the lymphocyte density gradient formulation. The PBMCs are then washed with mammalian cell culture medium such as RPMI 1640 medium.

To produce a stimulated reference standard sample (as opposed to an unstimulated negative control sample), the PMBC are usually immune-stimulated by stimulating mitogens with T cells at a temperature of 36-38 ℃ in an environment with a carbon dioxide content of 4-6%. In a preferred embodiment, a mixture of immunostimulants is used, said immunostimulants comprising phorbol (12-) myristate (-13-) acetate (PMA) (used at a concentration of 0.01-0.03. mu.g/ml, such as 0.02. mu.g/ml) and ionomycin (used at a concentration of 0.125-0.165. mu.g/mg, such as 0.145. mu.g/mg). This stimulation technique is suitable for cytokine production and if immune stimulated for 46 hours cytokine production can be detected in PMBC cells.

The PMBC is simultaneously bound to a commercially available extracellular protein transport inhibitor, such as brefeldin A or monensin, which results in the accumulation of cytokines in the PMBC. If brefeldin A is used, it is generally used at a concentration of 9.0-11.0. mu.g/ml, such as 10. mu.g/ml.

The stimulated PBMCs are then washed in a mixture of fetal calf serum ("FCS") and phosphate buffered saline ("PBS"), typically at a concentration of 9-12% v/v, e.g., 10%, and PBS typically at twice the concentration of FCS.

The washed PBMS were then resuspended in buffered culture (again including a double concentration of PBS and 9-12% FCS) with the addition of 0.1-20% (v/v) of a commercial transport fixative, 0.85% (w/v) PBS mixed with paraformaldehyde (0.1-0.2% w/v) and chromium chloride (0.5% w/v). One known example is the use of a fixative under the trade name "TransFix". The buffer culture solution and the fixative were used for the PBMC at a ratio of 8X 10 per ml⁶And (5) PBMCs.

For the production of unstimulated negative control cells, PBMCs were isolated from paraformaldehyde and chromium chloride immediately after fixation as described above, requiring storage at 4 ℃.

PBMCs (or standard samples for negative controls, fixed only) were cooled (+4 ℃) after washing after stimulation and fixation and freeze-dried in buffer (cryoprotectant). One commonly used buffer comprises twice the amount of 10% protein, usually fetal bovine serum or albumin in double concentration PBS. The use of the freeze-drying buffer helps to improve the stability of PBS during the freeze-drying process. These cells can be stored at low temperature (typically +4 ℃) prior to freeze-drying. For optimum and consistent reference standard sample quality, the freeze dryer shelves are pre-cooled so that the temperature is maintained at +4 ℃ during loading.

After fixation, cells from multiple donors can be mixed together to increase the overall yield of reference material. Cells from different donors were allowed to mix together during the fixation process without causing lymphocyte cross-reactivity. In addition, the immobilized stimulated cells may be mixed with immobilized unstimulated cells (which may also be used as a negative control) so that the population of cells produced demonstrates a positive control for the general level or minimal efficacy of cytokine stimulation.

The stimulated (or unstimulated) fixed cells were loaded into split/capped vials. A commercially available filling machine under the trade name Paxal is suitable for handling large numbers of samples.

After placing in the freeze-dryer, the PBMCs are typically freeze-dried for 5 days in one freeze-drying cycle. The residual moisture content after freeze-drying is typically 0.35%.

Once the fixative is washed off the stimulated cells, the PBMCs begin to leak out cytokines, although leakage is slow at low temperatures. Therefore, the dispensing and freeze-drying of PBMCs is completed within a few hours of fixative removal.

After freeze-drying, the lyophilized reference material can be stored for a longer period of time without degradation. The lyophilized sample can be reconstituted in twice the initial amount of distilled water. This is usually provided to the cells in single strength phosphate buffered saline.

In another embodiment of the invention, a cellular fraction enriched in a particular cell type is isolated from a mixed population of PMBC cells and subjected to stimulation and fixation as described above prior to remixing with fixed, unstimulated cells.

In this way, a population of cells can be produced with different levels of cytokines in a particular cell type, which can mimic a particular disease state. Specific cell types can be isolated by a variety of methods, such as flow cytometry and cell sorting, or using antibody-linked magnetic bead technology that has been commercially applied. These separation techniques can be used to isolate, for example, B cells, monocytes, natural killer cells, neutrophils, platelets, and the like, as well as specific subtypes, such as CD4 and CD8T cells. In addition, these differentially stimulated cell populations may be diluted with fixed, unstimulated cells, the resulting cell populations mimicking those found to be specific disease states.

The cellular reference standard sample prepared according to the method of the present invention can be reconstituted by adding water and used for cytokine analysis. These cells retain surface antigens as well as intracellular cytokines. Thus, these cells can be processed by staining techniques before being used in other analytical techniques and used as reference standard samples in e.g. flow cytometry analysis. Furthermore, these cells remain intact while still having the ability to release cytokines into their immediate environment, particularly when kept at higher temperatures such as 37 ℃, commonly used in enzyme linked immunosorbent assay (ELISPOT). Therefore, they can be used as reference standard samples in this type of analysis.

Storage tests showed that these lyophilized cells retain their properties after 5 months of storage, with evidence that they have a shelf life of up to many years, thus allowing them to be used as reproducible standard samples for long-term studies and diagnostics.

In addition to use as a reference standard, the lyophilized cells prepared by the methods described herein may also be used as a vaccine whose cell surface antigens are maintained in their intact state by the fixation and lyophilization processes.

Example 2 production of cell proliferation standards

By way of example, reference standard samples for Peripheral Blood Mononuclear Cell (PBMC) proliferation assays can be produced using the methods disclosed herein. In one embodiment of the invention, the "buffy coat" is obtained from a source such as a venous blood donor or a national blood collection facility. The buffy coat fraction of the blood is the fraction of the blood that is obtained by centrifugation and contains the majority of white blood cells and platelets. All of these buffy coats obtained were subjected to routine testing prior to use to ensure negativity for treponema pallidum hemagglutination test ("TPHA"), hepatitis b surface antigen detection ("HbsAg"), anti-human immunodeficiency virus 1 ("anti-HIV 1"), anti-human immunodeficiency virus 2 ("anti-HIV 2"), and anti-hepatitis c virus ("anti-HCV") prior to use.

Residual red blood cells present in the buffy coat can be removed by any standard method using a commercially available lymphocyte density gradient formulation, such as a lymphocyte separating agent, followed by separation/collection of peripheral blood mononuclear cells ("PBMCs") and then a second application of the lymphocyte density gradient formulation. The PBMCs are then washed in mammalian cell culture medium such as RPMI 1640 medium.

These PBMCs are then labeled with a dye such as carboxyfluorescein succinimidyl ester ("CFSE") or carboxyfluorescein succinimidyl ester ("CFDA-SE") that is commercially available. An example of a known dye is the dye sold under the trade name CellTrace CFSE Cell promotion Kit. The labeled cells were stimulated with the monoclonal antibody UCHT1 for 70-74 hours to induce proliferation prior to stabilization with fixative and freeze-drying or cryopreservation. The stimulation is generally carried out at ambient carbon dioxide levels of 4-6% and at temperatures of 3638 ℃. This stimulation induces cell proliferation. In another example, a mixture of immunostimulants is used, said stimulants typically comprising phorbol (12-) myristate (-13-) acetate (PMA) (used at a concentration of 0.01-0.03. mu.g/ml, such as 0.02. mu.g/ml) and ionomycin (used at a concentration of 0.125-0.165. mu.g/mg, such as 0.145. mu.g/mg). With such stimulation, proliferation usually occurs in less than 70-74 hours.

The PBMC are then washed in a mixture of fetal calf serum ("FCS") and phosphate buffered saline ("PBS") at a concentration of typically 9-12% v/v, for example 10%, and PBS at a concentration of typically twice the FCS concentration, according to the desired degree of proliferation (depending on the reference material to be produced).

The washed PBMS were then resuspended in buffered culture (again including a double concentration of PBS and 9-12% FCS) with the addition of 0.1-20% (v/v) of a commercial transport fixative, 0.85% (w/v) PBS mixed with paraformaldehyde (0.1-0.2% w/v) and chromium chloride (0.5% w/v). One known example is the use of a fixative under the trade name "TransFix". The buffer medium and the fixative were used in the PBMC at a ratio of 8X 10⁶1ml of PBMC was used.

After fixation, the PBMCs were washed, cooled (+4 ℃) and freeze-dried in buffer (cryoprotectant). Commonly used buffers include twice the amount of 10% protein, usually fetal bovine serum or albumin in double concentration PBS. The use of the freeze-drying buffer helps to improve the stability of PBS during the freeze-drying process. These cells can be stored at low temperature (typically +4 ℃) prior to freeze-drying. For optimum and consistent reference standard sample quality, the freeze dryer shelves are pre-cooled so that the temperature is maintained at +4 ℃ during loading.

The labeled proliferating fixed cells are then loaded into subpackages/capped vials. A commercially available filling machine under the trade name Paxal is suitable for handling large numbers of samples.

After placing in the freeze-dryer, the PBMCs are typically freeze-dried for 5 days in one freeze-drying cycle. The residual moisture content after freeze-drying is typically 0.35%.

By freeze-drying, the lyophilized reference material can be stored for a longer period of time without degradation. The lyophilized sample can be reconstituted in twice the initial amount of distilled water. This is usually provided to the cells in single strength phosphate buffered saline.

In another embodiment of the invention, the cell fraction is enriched with specific cell types isolated from a mixed population of PMBCs, and the cell fraction is subjected to labeling, proliferation and fixation as described above. In this way, cell populations of varying degrees of proliferation can be generated in a given cell type. Specific cell types can be isolated by a variety of methods, such as flow cytometry and cell sorting, or using antibody-linked magnetic bead technology that has been commercially applied. These separation techniques can be used to isolate, for example, B cells, monocytes, natural killer cells, neutrophils, platelets, and the like, as well as specific subtypes, such as CD4 and CD8T cells. In addition, these differentially proliferating cell populations may be diluted with labeled non-proliferating fixed cells, thereby generating cell populations that mimic those specific disease states that have been discovered.

The reference standard sample of cells prepared according to the method of the present invention can be reconstituted by adding water and used as a reference standard sample for cell proliferation analysis. These cells retain surface antigens as well as intracellular cytokines. Thus, these cells can be processed by staining techniques of the art and used as reference standard samples in, for example, flow cytometry analysis.

Storage tests showed that these lyophilized cells retain their properties after 5 months of storage, with evidence that they have a shelf life of up to many years, thus allowing them to be used as reproducible standard samples for long-term studies and diagnostics.

Claims (33)

1. A method of preparing a cellular reference material comprising the steps of:

(a) obtaining a cell sample isolated from a human or animal body, said cell sample comprising cells selected from the group consisting of:

peripheral blood mononuclear cells;

a cell line;

a thrombocyte;

(b) stimulating the production of cytokines by the cells in the presence of an inhibitor of cytokine secretion;

(c) fixing said stimulated cells with a fixative;

(d) freeze-drying the above fixed stimulated cells.

2. A method of preparing a cellular reference material comprising the steps of:

(i) obtaining a mixed population of cells isolated from a human or animal body, said mixed population of cells comprising a plurality of cell types selected from the group consisting of:

peripheral blood mononuclear cells;

a thrombocyte;

(ii) fractionating said mixed population of cells to produce a plurality of fractions, each fraction containing a different population of cell types;

(iii) subjecting one or more of the above components to cellular stimulation in the presence of an inhibitor of cytokine secretion to produce cytokines;

(iv) adding a fixative to fix the cells in each fraction;

(v) combining the above components to make a differentially stimulated cell mixture;

(vi) the differentially stimulated cell mixture is stored in a freeze-dried or ultra-low temperature.

3. A method according to claim 1 or claim 2, wherein the cell sample is isolated from a mammalian body.

4. The method of claim 3, wherein the cell sample is isolated from a human.

5. A method according to any preceding claim, wherein the cells are stimulated by the addition of a mitogen.

6. The method of claim 5, wherein the mitogen comprises PMA (phorbol 12-myristate 13-acetate).

7. The method of claim 5, wherein said mitogen comprises ionomycin.

8. The method of claim 5, wherein the mitogen comprises a mixture of PMA (phorbol 12-myristate 13-acetate) and ionomycin.

9. The method of any preceding claim, wherein the cytokine secretion inhibitor comprises brefeldin a.

10. A method according to any preceding claim, wherein the fixing agent comprises paraformaldehyde.

11. A process according to claim 10 wherein the fixing agent comprises a mixture of paraformaldehyde and chromium chloride.

12. The method of any preceding claim, further comprising the step of removing residual fixative after fixing the cells and prior to preserving the cells.

13. A method according to any preceding claim, further comprising the step of exposing said fixed cells to a hypertonic environment prior to preserving said cells.

14. A method according to any preceding claim, additionally comprising the step of adding a cryoprotectant to the cells prior to preservation.

15. A method of preparing a cellular reference material comprising the steps of:

obtaining a plurality of cell samples isolated from a plurality of individuals;

treating each cell sample according to the method of any preceding claim when dependent on claim 1;

before preserving said cells, additionally comprising a step of combining fixed cells derived from said plurality of samples.

16. A method of preparing a cellular reference material comprising the steps of:

obtaining a plurality of mixed cell populations isolated from a plurality of individuals;

treating each mixed cell population according to the method of any preceding claim when dependent on claim 2;

further, the method may further comprise a step of combining fixed cells derived from a plurality of the mixed cell groups before the cells are preserved.

17. A method of preparing a cellular reference material comprising the steps of:

preparing non-stimulated fixed cells according to the method steps of any one of the preceding claims, but omitting the stimulating step (b) or (iii);

combining said unstimulated fixed cells with stimulated fixed cells prepared according to the method steps of any one of the preceding claims;

the mixed cells are preserved by freeze drying or ultra-low temperature method.

18. A method of preparing a cellular reference material comprising the steps of:

(i) obtaining a mixed population of cells isolated from a human or animal body, said mixed population of cells comprising a plurality of cell types selected from the group consisting of: peripheral blood mononuclear cells, thrombocytes;

(ii) fractionating said mixed population of cells to produce a fraction, said fraction being predominantly of a single cell type;

(iii) labeling cells of the fraction;

(iv) fixing the labeled cells in the above-mentioned fraction by adding a fixing agent;

(v) the labeled cells were stored by freeze-drying.

19. A method of preparing a cellular reference material comprising the steps of:

(a) obtaining proliferation-competent mammalian cells isolated from a human or animal body;

(b) labeling said cells to differentiate progeny cells in the process of cell proliferation;

(c) stimulating said labeled cells to proliferate;

(d) allowing said cells to proliferate;

(d) fixing the proliferated cells by adding a fixing agent;

(e) the resulting cells are stored in a freeze-dried or ultra-low temperature.

20. The method of claim 19, wherein the cell is isolated from a mammalian body.

21. The method of claim 20, wherein the cell is isolated from a human.

22. The method of any one of claims 18 to 21, wherein the cells are stimulated by the addition of a mitogen.

23. The method of claim 22, wherein the mitogen comprises PMA (phorbol 12-myristate 13-acetate).

24. The method of claim 22, wherein said mitogen comprises ionomycin.

25. The method of claim 22, wherein the mitogen comprises a mixture of PMA (phorbol 12-myristate 13-acetate) and ionomycin.

26. A method according to any one of claims 19 to 25 wherein the fixing agent comprises paraformaldehyde.

27. The method of claim 26 wherein the fixative comprises a mixture of paraformaldehyde and chromium chloride.

28. The method of any one of claims 19 to 27, further comprising the step of removing residual fixative after fixing said cells and prior to preserving said cells.

29. The method of any one of claims 19 to 28, further comprising the step of exposing said fixed cells to a hypertonic environment prior to preserving said cells.

30. The method of any one of claims 19 to 29, further comprising the step of adding a cryoprotectant to the cells prior to preserving said cells.

31. The method of any one of claims 19-30, wherein the cells are labeled with a fluorescent label.

32. A method according to claim 31, wherein said fluorescent label comprises CFSE (carboxyfluorescein succinimidyl ester).

33. A method of preparing a cellular reference material substantially as described herein.