Device for collecting, preserving and storing eukaryotic cells contained in a sample of biological fluid for further cell analysis

Abstract

A device for collecting, preserving and storing a sample of biological fluid comprising a porous medium for collecting and maintaining the membrane integrity of the eukaryotic cells contained in the sample, and their constituents is disclosed, the medium comprising at least 80% by weight of artificial components, advantageously at least 85%, preferably at least 90% of a total weight of the medium; and having a mean flow pore size (MFP) of at least 3 μm.

Claims

1. A device for collecting, preserving and storing a sample of biological fluid comprising, the device comprising a porous medium for collecting and maintaining the membrane integrity of the eukaryotic cells contained in the sample, and their constituents, the medium comprising at least 80% by weight of artificial components, advantageously at least 85%, preferably at least 90% of a total weight of the medium; and having a mean flow pore size (MFP) of at least 3 μm.

2. The device according to claim 1, characterized in that the artificial component is chosen from the group comprising artificial fibers and artificial foams.

3. The device according to any one of claim 1 or 2, characterized in that the medium is a nonwoven.

4. The device according to any one of the preceding claims, characterized in that the artificial component comprises: semisynthetic fibers advantageously chosen from the group comprising: rayon fibers, lyocell®, fibers, viscose fibers, and mixtures thereof; and/or synthetic fibers advantageously chosen from the group comprising: polyethylene terephthalate (PET) fibers, glass fibers, glass microfibers, and their mixtures.

5. The device according to any one of the preceding claims, characterized in that the medium comprises up to 15% by weight of a binder, advantageously up to 10%.

6. The device according to any one of the preceding claims, characterized in that the binder is chosen from the group comprising polyvinyl alcohol (PVOH), advantageously in the form of fibers, and latexes, advantageously latexes of acrylic styrenes.

7. The device according to any one of the preceding claims, characterized in that the medium has an MFP of between 10 μm and 200 μm, advantageously between 30 μm and 180 μm, preferably between 45 μm and 150 μm.

8. The device according to any one of claims 4 to 7, characterized in that the semi-synthetic fibers and/or the synthetic fibers have a moisture regain value of less than approximately 5%.

9. The device according to any one of claims 4 to 8, characterized in that the semi-synthetic fibers and/or the synthetic fibers have a contact angle with water of less than approximately 75°, advantageously less than approximately 60°.

10. The device according to any one of claims 4 to 9, characterized in that the semi-synthetic fibers and/or the synthetic fibers have a diameter of at least 3 μm.

11. The device according to any one of claims 1 to 4, characterized in that the medium contains exclusively artificial components, preferably semi-synthetic fibers and/or synthetic fibers.

12. The device according to any one of the preceding claims, characterized in that the medium is impregnated with an isotonic preservative and optionally at least one preservative and/or at least one anticoagulant agent.

13. The device according to any one of the preceding claims, characterized in that it comprises at least one physical and/or chemical means capable of limiting the evaporation of the biological sample, advantageously a film impermeable to gases and liquids.

14. The device according to any one of the preceding claims, characterized in that the medium has a thickness between 100 μm and 2500 μm, advantageously between 200 μm and 1200 μm.

15. The device according to any one of the preceding claims, characterized in that the medium is sandwiched between two supports, the two supports being perforated the one facing the other to reveal the medium.

16. The device according to claim 15, characterized in that one of the supports is covered with a film impermeable to gases and liquids, at least in the region opposite the medium, while the other support is extended laterally by a flap covering at least the area of the support facing the medium, preferably the entire surface of the support.

17. A method for collecting, preserving and storing a sample of biological fluid for cell analysis, comprising a step of depositing a sample of said biological fluid on the medium of the device according to any one of claims 1 to 16.

18. The method according to claim 17, characterized in that it comprises a step of impregnating the medium with an isotonic preservation buffer and optionally at least one preservative and/or at least one anticoagulant agent, advantageously before, or during, or after the collecting of the biological fluid sample, preferably after collecting the biological fluid sample.

19. The method according to any one of claims 17 to 18, characterized in that it comprises a subsequent step of recovering the cells stored on the medium by elution.

20. The method according to any one of claims 17 to 19, characterized in that it comprises a subsequent step of analysis of the cells and/or the constituents of the cells contained in the sample.

21. The method according to any one of claims 17 to 20, characterized in that the sample is a blood sample.

22. The method according to any one of claims 17 to 21, characterized in that the constituents are chosen from: a. Organelles such as: i. Core, ii. Mitochondria, iii. Lysosomes iv. Exosomes b. Nucleic acids such as: i. Messenger ribonucleic acid (mRNA), ii. micro-RNA (miRNA), iii. Circular deoxyribonucleic acid (DNA), iv. Long non-coding RNA, v. Mitochondrial DNA, vi. Unfragmented DNA, vii. Intact chromosomes, c. Proteins and Molecules such as: i. Hemoglobins (HBs), ii. Cytoplasmic molecules, iii. Ribosomes, d. Intracellular parasites such as: i. Plasmodium, Leishmania, ii. Intracellular bacteria, e. and all intracellular molecules or all interesting organelles, whether endogenous or exogenous.

Description

DESCRIPTION OF THE FIGURES

[0131] The manner of carrying out the invention, as well as the advantages which result therefrom, will emerge clearly from the description of the embodiment which follows, with the support of the appended figures.

[0132] FIG. 1 shows an exploded perspective view of a collection device according to the invention.

[0133] FIG. 2 represents the method of taking and collecting samples for cell analysis implementing the device according to FIG. 1.

[0134] FIG. 3 is a photograph by phase contrast microscopy of the sample obtained by means of the device according to FIG. 1 implemented in the method of FIG. 2.

[0135] FIG. 4 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific DAPI and CD45 markers, of a biological sample obtained by means of the device of the FIG. 1 implemented in the method of FIG. 2.

[0136] FIG. 5 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific markers CD3 and CD19, of a cell population.

[0137] FIG. 6 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific CD4 and CD8 markers, of a cell population.

[0138] FIG. 7 represents a two-dimensional diagram obtained after an analysis by flow cytometry, on the basis of the expression of the specific EpCAM and CD45 markers, of a cell population.

[0139] FIG. 8 shows, in table form, the characteristics of examples of media according to the invention and counter-examples.

EXAMPLES

Example 1: Example of a Device According to an Aspect of the Invention

[0140] FIG. 1 illustrates a particular embodiment of a device according to an aspect of the invention in the form of a card (1), comprising a porous medium in the form of a nonwoven (2).

[0141] The nonwoven medium (2) is sandwiched between two identical supports (3) and (4) made of cardboard. The supports (3) and (4) respectively have windows (31) and (41). These windows are placed opposite one another.

[0142] The presence of windows on the support (3) allows access to the nonwoven medium (2) for deposit of the sample of biological fluid. The presence of windows makes it possible to recover the sample deposited on the nonwoven medium (2) by punching or by creep.

[0143] An adhesive film (5) impermeable to gases and liquids is bonded to the external face of the support (4). A flap (6) is intended to be folded over the external face of the support (3) after depositing the biological sample.

[0144] The flap (6) allows, after deposit of the sample, to enclose the nonwoven medium (2) between the adhesive film (5) and the flap (6). Thus the nonwoven medium (2) and the biological sample are in a confined atmosphere isolated from air, light, and ambient humidity.

Example 2: Implementation of the Collection Device and Cell Analysis

a) Application to a Blood Sample

[0145] As shown in FIG. 2, there is shown a method comprising several steps, namely:

[0146] Step 1: Collection of the blood sample using a medium as described in Example 1, for example.

[0147] Step 2: Storage of the device at room temperature, that is approximately 23° C.

[0148] Step 3: Recovery of the sample by cutting out of a disc of the medium placed in a tube.

[0149] Then, the cells are eluted, that is to say recovered and put in solution using an isotonic buffer, the PBS. This operation is carried out after 24 hours of storage.

[0150] Step 4 (optional, depending on the nature of the biological sample and the type of subsequent cell analysis): Centrifugation of the eluates for 1 to 5 minutes between 300 and 600 g.

[0151] In the case where the sample does not contain intact cells, all the cell membranes are lysed, that is to say that the membrane integrity is not preserved. Centrifugation does not make it possible to obtain a cell pellet but only debris of cell constituents in suspension. In the case of a sample containing cells which have preserved their membrane integrity, the centrifugation will form a pellet consisting of the intact cells which can then be recovered for analysis. In other words and in this example, the preservation of the membrane integrity of the cells obtained from a blood sample is evaluated by centrifugation.

[0152] Step 5: Cell analysis performed on the cell pellet obtained in step 4.

[0153] In this step, the cells recovered in Step 4 are suspended to be analyzed, for example by flow cytometry.

[0154] This technique makes it possible to sort the cell populations via the identification of fluorescence signals: [0155] Fluorescence emitted by the cell itself (autofluorescence); [0156] Fluorescence emitted by the antibody, coupled to a fluorochrome, which specifically binds to the cell.

[0157] Morphological parameters of size and granularity can also be measured and exploited by flow cytometry.

[0158] As shown in FIG. 8, the media 1 to 11, having the characteristics according to the invention, make it possible to preserve the membrane integrity of the blood cells for 24 hours after collection, on a wet (that is to say impregnated with isotonic preservation buffer) or dry medium (that is to say not impregnated with isotonic preservation buffer) and after storage at 4° C. or at room temperature (23-25° C.).

[0159] In addition, when the media 1 to 11 are impregnated with an isotonic preservation buffer, the preservation of the membrane integrity of the cells of the sample extends up to 72 hours after their collection and storage at 4° C. or at room temperature (23-25° C.).

[0160] The medium 12 makes it possible to preserve the membrane integrity of the cells of the sample after 24 hours of storage at 4° C. or at room temperature in a humid environment, and after 24 hours of storage at 4° C. in a dry environment.

[0161] The media 13 and 14 (counter-examples) do not have the characteristics according to the invention (these media contain 100% natural fibers, by weight). The results show that these media when used in dry conditions, that is to say without impregnation with an isotonic preservation buffer, do not allow the membrane integrity of the cells to be preserved after 24 hours of storage at 4° C. or at room temperature (23-25° C.).

[0162] The media 15 and 16 (counter-examples) do not have the characteristics according to the invention. In particular, these media have an MFP of less than 3μm (respectively 2.2 μm and 2.9 μm), not allowing the membrane integrity of the cells of a blood sample to be preserved, whatever the collection, preservation and storage conditions. Accordingly, the MFP is a key parameter in order to preserve the membrane integrity of the cells.

[0163] The determination of the survival of the cells of the blood sample was evaluated after 72 hours of storage in a dry medium, at 4° C. or at room temperature.

[0164] The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Composition 72 hours DRY (in % by total Thickness ENVIRONMENT weight of the (μm) at MFP Room medium) 100 Kpa (μm) 4° C. temperature Medium 100% PET 110 70 Yes Yes No. 1 Medium 91% of 324 119 Yes Yes No. 2 glass fiber + 9% PVOH

[0165] The results of Table 1 show that the membrane integrity is preserved, at least for media 1 and 2, after 72 hours of storage at 4° C. or at room temperature, in a dry medium, that is to say without adding isotonic conservation buffer.

b) Analysis of Results

[0166] The cell pellet obtained at the end of step 4 mentioned above contains in particular red blood cells (erythrocytes) and white blood cells (leukocytes).

[0167] Red cells are anucleate eukaryotic cells, that is to say cells devoid of a nucleus following its expulsion during the differentiation of the erythrocyte lineage, as mentioned previously. They have a characteristic concave shape which allows their identification via phase contrast microscopy.

[0168] Leukocytes have a nucleus, possibly multilobed. The presence of this nucleus then induces a change in phase of the light wave which makes it possible to identify them. The results are shown in FIG. 3.

[0169] The dark cells correspond to red blood cells and the white cells to leukocytes. This photograph confirms that the cells have retained their membrane integrity, all the way to their shape, as shown by the biconcavity that is characteristic of red blood cells, thus making it possible to analyze them.

[0170] These visual results are confirmed by an analysis of the sample by flow cytometry, either by autofluorescence or by binding to particular antibodies (anti-CD8, anti-EpCAM, etc.).

[0171] This type of identification requires the use of specific markers expressed on the surface of the cells, which implies that the membrane integrity is preserved.

[0172] The results obtained after analysis of the sample by flow cytometry are presented in the form of a 2-dimensional diagram.

[0173] As shown in FIG. 4, the identification and the analysis or the cell sorting are carried out on the basis of the expression of the specific DAPI (4′,6-diamidino-2-phenylindole) and CD45 (“cluster of differentiation 45” or differentiation group) markers present on the surface of cells.

[0174] Thus, the cells emitting a weak fluorescence corresponding to the DAPI signal as well as a weak fluorescence corresponding to the CD45 signal (DAPI.sup.− and CD45.sup.−) are anucleate cells (no DAPI labeling), which are not leukocytes (no CD45 marking). These cells are identified as corresponding to red blood cells.

[0175] Similarly, the DAPI.sup.− and CD45.sup.+ cells are living leukocytes and the DAPI.sup.+ and CD45.sup.+ cells are dead leukocytes but which have kept their membrane integrity.

[0176] After the identification and collection of these different cell types and using multiplexing, the previously identified living leukocytes are analyzed to identify the subpopulations (FIG. 5): [0177] CD3.sup.− and CD19.sup.+: B lymphocytes; and [0178] CD3.sup.+ and CD19.sup.−: T lymphocytes. After the identification and collection of these different cell types, the T lymphocytes are analyzed to identify the subpopulations (FIG. 6): [0179] CD4.sup.− and CD8.sup.+: T8 lymphocytes; and [0180] CD4.sup.+ and CD8.sup.−: T4 lymphocytes.

[0181] The different cell populations present in a sample obtained using the device according to the invention can be identified by flow cytometry. FIG. 7 represents the identification of red blood cells, leukocytes and cancer cells depending on whether the cells express the EpCAM (“Epithelial cell adhesion molecule”) and CD45 biomarkers, in a blood sample obtained by means of the device according to the invention.

[0182] Thus, the populations present in the sample are: [0183] EpCAM.sup.− and CD45.sup.−: red blood cells; [0184] EpCAM.sup.− and CD45.sup.−: leukocytes; and [0185] EpCAM.sup.+: cancer cells.

[0186] As these results show, the device of the invention makes it possible to collect, preserve and store, thanks to the media which it contains, a sample of biological fluid under conditions such that the membrane integrity of the cells is preserved, thus making further cell analysis possible.