Method for detecting potentially viable microorganisms present in a sample of a cell product

Abstract

The present invention relates to a method for isolating potentially viable microorganisms present in a sample comprising 106 to 108 immune cells or their precursor cells, the method comprising the following steps: - the step of contacting said sample with the following: a lysis composition comprising: o a lysis buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume; o a lysis solution containing a saponin at a concentration of 0.03% to 4% of the reaction volume; an endonuclease capable of degrading nucleic acids released by the action of said lysis composition; an endopeptidase acting at a pH of 7 to 8; - the step of isolating potentially viable microorganisms present in said sample by filtering said sample through a filter having a pore diameter of 0.30 to 0.50 μm. The present invention also relates to a related solid-phase cytometry detection method.

Description

Technology Field

[0001] The present invention relates to the field of microbiological management technology in the pharmaceutical industry. The present invention relates particularly to the preparation of cell product samples for detecting viable microorganisms, and particularly to detection by solid-phase cytometry. Background Technology

[0002] Advanced Therapy Medicinal Products (ATMPs) are a type of pharmaceutical product currently undergoing rapid expansion. While currently limited to specific targeted applications (hematological and rare diseases), they are expected to occupy an increasingly important position in the healthcare system as their efficacy is now proven (in 2020, over 1,000 clinical trials were conducted, nearly 100 of which were Phase III).

[0003] These drugs are based on the use of cell products (autologous, allogeneic, or xenogeneic) that have undergone substantial manipulation in vitro to alter biological properties, physiological functions, or structural properties before being administered to humans.

[0004] Nevertheless, the large-scale production and use of these medicines have faced numerous difficulties, particularly the following:

[0005] - Much higher production costs compared to chemical pharmaceuticals (new and complex production processes, low production volume, etc.)

[0006] - The very short stability (approximately a few hours) of finished products used in a fresh state and the resulting need to perform quality control within a very short time.

[0007] - Since these medicines cannot be sterilized, the risk of microbial contamination increases.

[0008] Microbiological control is a critical step in the ATMP production cycle, particularly regarding sterility testing, which is a prerequisite for drug release. This is described in Section 2.6.27 of the European Pharmacopoeia, “Microbiological examination of cell-based preparations.” Currently, time and cost constraints associated with such microbiological control are one of the major factors hindering the development of the ATMP industry.

[0009] The European Pharmacopoeia mandates sterility testing for finished products as a prerequisite for release. Conventional methods, namely cell culture in media and visual inspection, require a minimum of 14 days to ensure a non-contamination state. Additionally, alternative methods based on cell culture but featuring automated reading capabilities are approved by the Pharmacopoeia and require a culture period of at least 7 days, and more generally 10 days, to yield results. Currently, bioMerieux’s BACT / ALERT® solution, which detects microbial growth induced by carbon dioxide (CO2) generation, serves as the reference solution for ATMP sterility control.

[0010] However, these methods can affect the patient's life due to particularly long response times. Additionally, these methods often require the collection of relatively large sample volumes, which are frequently incompatible with the volumes produced in the context of ATMP manufacturing activities, and even more so in autologous situations.

[0011] With the aim of reducing the time required and improving detection capabilities, other technologies are currently being researched to perform sterility testing on ATMPs:

[0012] - Quantification of ATP (adenosine triphosphate)

[0013] - Molecular biological test.

[0014] Another technique not yet considered for application in sterility testing for ATMP is solid-phase cytometry.

[0015] Solid-phase cytometry enables the very rapid quantification of microorganisms, such as bacteria, yeast, or fungi, within a sample. The sample is filtered through a membrane, and all microorganisms present in the sample are recovered onto the membrane. Subsequently, viable microorganisms penetrate into the microorganisms and are cleaved by esterases to release fluorophores (fluorescein) into the microorganisms. These fluorophores are then specifically labeled using molecules that cause them to emit light at a specific wavelength. A laser beam scans the entire surface of the membrane to excite the fluorophores, thereby enabling the detection and counting of viable microorganisms that may be present in the sample.

[0016] The use of solid-phase cytometry in ATMP sterility testing presents numerous challenges. The main limitations of this technique are 10 5 It lies in the characteristics of the lysed target cells, particularly including immune cells and their precursor cells, with cell / ml or higher cell concentrations.

[0017] The problem of such high cell concentrations also occurs when bacteria are detected in blood products, namely, generally whole blood. Accordingly, WO03025207 discloses a method for concentrating bacteria in a blood product, comprising reducing the concentration of a blood cell population by the selective agglutination of said cells. A filtration step allows non-agglutinous pathogenic microorganisms to be collected in the filtrate and blood cell aggregates to be retained on the filter. The method then optionally includes a step of passing the remaining cells through the filter again after selective lysis.

[0018] The step of reducing the concentration of the blood cell population is approximately 4 log(10) with respect to platelet concentration. 9 cells / ml to 10 5 cells / ml), approximately 5 log(10) for red blood cell concentration 10 cells / ml to 10 5 It enables a reduction in cells / ml. Although microorganisms are smaller than aggregated blood cells, there is a risk of retention, which can lead to microbial loss and result in false negatives in detection methods.

[0019] The problem of high cell concentrations also arises in blood samples for other detection methods, such as those in the field of molecular biology. Patents EP2718713 and EP2510123 disclose a method for selectively lysing blood cells in samples that may contain microorganisms, using a controlled lysis buffer to recover intact, dead, or viable microorganisms. The DNA of the microorganisms is then extracted for detection by PCR. In molecular biological detection, microbial viability is not an essential criterion. Therefore, a disadvantage of these methods is that the lysis buffer does not guarantee the preservation of microbial viability, whereas this is required for detection by solid-phase cytometry with excellent sensitivity.

[0020] Zelenin (Biotechnol Lett 2015 37: 825-830) describes a protocol for isolating viable bacteria in blood samples by using saponin at a concentration of 0.5% to 2.5% to destroy red blood cells, followed by osmotic shock to lyse white blood cells. A disadvantage of this method is that it cannot be applied to samples containing high concentrations of immune cells such as white blood cells, which are much more difficult to lyse than red blood cells. After this isolation method, molecular biological detection and antibiotic susceptibility testing are performed.

[0021] EP3063290 discloses a method for isolating microorganisms from a sample having a high cell density, comprising the use of a lysis buffer containing 0.05 to 0.5% SDS and Benzonase® nucleases. The sample contains 10 within at least 5 ml of the sample. 6 Pieces up to 5×10 8 It contains cells. Afterwards, the microorganisms are lysed and detected by molecular biology. This method has the disadvantage that it cannot preserve the viability of microorganisms by using anionic detergents such as SDS.

[0022] In addition, EP2601304 describes the preparation of blood samples for identification by mass spectrometry. For this purpose, human-derived particles must be lysed with saponin, and centrifugation or microfiltration must be performed to recover only viable microorganisms. These microorganisms are then cultured, separated from the culture medium, and identified by mass spectrometry. After the centrifugation step, although containing potential microorganisms, there are also 5 × 10 per milliliter of blood 9A 1% saponin solution is added to a precipitate containing 10 red blood cells, 7,000 white blood cells, and 50,000 platelets. The mixture is then centrifuged again to recover the precipitate. This method is not suitable for samples containing high concentrations of immune cells such as white blood cells, as these cells are much more difficult to lyse than red blood cells.

[0023] Another challenge when applying ATMP lies in the filterability of the cell matrix. Therefore, one solution lies in preparation methods that enable the sample to be filterable. Nevertheless, such methods can irreversibly alter the microbial load and may either completely eliminate the microorganisms or damage them to the point where viability markers can no longer detect them. Accordingly, WO2019051272 discloses the preparation of highly viscous and non-filterable samples for detection by solid-phase cytometry. The use of isopropyl myristate-based solvents does not resolve the problems faced with samples having high cell concentrations.

[0024] Similarly, the paper in Journal of Microbiological Methods 64 (2006) 420-423 discusses the presence of in respiratory secretions Aspergillus fumigatus The cytometric detection of [the sample] is described, which is difficult to filter. The proposed pretreatment involves the addition of rhDNAse I, trypsin, HEPES buffer, and DTT / Triton X-100. The use of this pretreatment, which is specifically adjusted for highly viscous samples, does not solve the problems faced with samples having high cell concentrations.

[0025] Therefore, there is a need to develop a method for detecting viable microorganisms from difficult-to-filter samples containing high concentrations of immune cells or their precursor cells.

[0026] One of the objectives of the present invention is to propose a method for isolating viable microorganisms that may be present in extremely small quantities within a sample, while simultaneously removing immune cells or their precursor cells that are present in very large amounts.

[0027] Accordingly, the first aspect of the present invention is 10 6 to 10 8 The present invention relates to a method for isolating viable microorganisms potentially present in a sample containing immune cells or their precursor cells, said method comprising the following steps:

[0028] - Step of bringing the sample into contact with the following:

[0029] A lysing composition comprising the following

[0030] o lysis buffer containing a nonionic detergent at a concentration of 0.004% to 0.050%

[0031] o and / or a lysis solution containing saponin at a concentration of 0.03% to 4%

[0032] Endonuclease capable of degrading nucleic acids released by the action of the above-mentioned dissolution composition

[0033] Endopeptidase acting at pH 7 to 8,

[0034] - A step of filtering potentially viable microorganisms through a filter having a pore diameter of 0.30 to 0.50 μm, that is, a step of separating potentially viable microorganisms within a sample by filtering the sample through a filter having a pore diameter of 0.30 to 0.50 μm.

[0035] The present invention is preferably 10 6 to 10 8The present invention relates to a method for isolating viable microorganisms potentially present in a sample containing immune cells or their precursor cells, said method comprising the following steps:

[0036] - Step of bringing the sample into contact with the following:

[0037] A lysing composition, wherein the lysing composition is a lysing buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume and a saponin at a concentration of 0.03% to 4% of the reaction volume.

[0038] An endonuclease capable of degrading nucleic acids released by the action of the above-mentioned dissolution composition

[0039] Endopeptidase acting at pH 7 to 8,

[0040] - A step of filtering potentially viable microorganisms through a filter having a pore diameter of 0.30 to 0.50 μm, that is, a step of separating potentially viable microorganisms within a sample by filtering the sample through a filter having a pore diameter of 0.30 to 0.50 μm.

[0041] Accordingly, the present invention effectively lyses immune cells or their precursor cells present at very high concentrations while maintaining viable microorganisms, thereby enabling them to be detected by a method that requires the microorganisms to maintain a viable state. The immune cells are preferably CAR-T cells.

[0042] One of the advantages of the present invention is that it enables effective and rapid preparation of samples. The time required to filter the sample after contacting it with the lysis composition, endonuclease, and endopeptidase is less than 30 minutes, preferably less than 20 minutes. The method according to the present invention allows the viability of microorganisms to be maintained for detection and, in particular, enables detection by solid-phase cytometry, which is a very rapid detection method. The efficiency of lysis according to the present invention is attributed particularly to the synergy between the lysis composition, endopeptidase, and endonuclease. In fact, the lysis step by the lysis composition causes a loss of viability in immune cells or their progenitor cells. This acts by structurally disrupting the cell's plasma membrane and increasing its permeability by forming complexes with lipids within the cell membrane. This mechanism subsequently causes endopeptidase to hydrolyze membrane proteins, thereby rupturing the cell. The action of endopeptidase also continues, breaking down the nuclear membrane and releasing intracellular substances. Endonuclease further enhances lysis by degrading nucleic acids.

[0043] The above endopeptidase is preferably trypsin.

[0044] The above nonionic detergent is preferably a polyoxyethylene detergent such as BRIJ or NP40.

[0045] When high concentrations of cells are present, filtering the sample is difficult, and therefore the efficiency of lysis becomes a key factor. The small debris produced by the lysis can prevent the filter from clogging, which can interfere with the detection of microorganisms. In the case of detection by solid-phase cytometry, effective lysis prevents the presence of a significant level of background fluorescence noise that can mask the presence of microorganisms.

[0046] Another objective of the present invention is to enable rapid detection by solid-phase cytometry. This detection takes 4 to 6 hours. Accordingly, another aspect of the present invention is 10 6 to 10 8 The present invention relates to a method for detecting microorganisms potentially present in a sample containing immune cells or their precursor cells, said method comprising the following steps:

[0047] - Step of performing the separation method according to the present invention

[0048] - A step of labeling viable microorganisms retained on a porous membrane using a viability stain

[0049] - A step of scanning the porous membrane using a laser beam or a camera

[0050] - A step of determining the presence of surviving microorganisms captured in the above membrane.

[0051] Since immune cells or their precursor cells are labeled as microorganisms by viability stains, the complete lysis of immune cells can prevent these cells from being labeled, thereby reducing false positives. Additionally, lysis yields very small cell debris, which prevents microorganisms from attaching to these debris and consequently prevents the microorganisms from being masked. Therefore, this method reduces false negatives.

[0052] Another aspect of the present invention relates to a lysis kit comprising the following:

[0053] - Dissolution buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume

[0054] - A solution containing saponin at a concentration of 0.03% to 4% of the reaction volume

[0055] - Endonuclease

[0056] - Endopeptidase acting at pH 7 to 8.

[0057] The lysis kit according to the present invention is non-toxic to microorganisms and enables effective lysis of immune cells or their precursor cells. Specific details for implementing the invention

[0058] The present invention relates to a method for isolating viable microorganisms potentially present in a sample containing a large amount of immune cells or their precursor cells, and for detecting said microorganisms using solid-phase cytometry.

[0059] The first aspect of the present invention is 10 6 to 10 8 The present invention relates to a method for isolating viable microorganisms potentially present in a sample containing immune cells or their precursor cells, said method comprising the following steps:

[0060] - Step of bringing the sample into contact with the following:

[0061] o A lysing composition comprising the following

[0062] A lysis buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume

[0063] and / or a lysis solution containing saponin at a concentration of 0.03% to 4% of the reaction volume

[0064] o Endonuclease capable of degrading nucleic acids released by the action of the above-mentioned dissolution composition

[0065] o Endopeptidase acting at pH 7 to 8,

[0066] - A step of isolating potentially viable microorganisms present in a sample by filtering the sample through a filter with a pore diameter of 0.30 to 0.50 μm.

[0067] Preferably, the dissolution composition is a dissolution buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume and a saponin at a concentration of 0.03% to 4% of the reaction volume.

[0068] The sample according to the present invention is 10 6 to 10 8 It is a clinical sample comprising immune cells or their progenitor cells. In a preferred embodiment, the number of cells in the sample is 10 6 to 10 7 It is a dog's immune cell or its precursor cell, more preferably 10 6 to 10 7 It is a cell. The amount of sample used depends on the source of the sample. In a preferred embodiment, the sample volume is 100 μl to 2 ml, more preferably 500 μl to 1.5 ml.

[0069] The above immune cells or their progenitor cells belong to advanced therapy medicinal products or cell therapy products. The above immune cells may be selected from NK cells, monocytes, B lymphocytes, and T cells, and the T cells may be natural or genetically modified, such as regulatory T cells, tumor-infiltrating T cells, cytotoxic T cells, helper T cells, and T cells having a chimeric antigen receptor (CAR).

[0070] NK cells (or NK lymphocytes) are cells of the innate immune system. They are non-T (CD3-) lymphocytes and non-B (CD19-) lymphocytes, and in humans, they are characterized by CD56, CD16, and NK markers.

[0071] Monocytes are leukocytes that differentiate into macrophages, dendritic cells, or osteoclasts.

[0072] B lymphocytes are immune cells responsible for antibody production.

[0073] Regulatory T cells are a subpopulation of CD4+ T cells that inhibit the proliferation of other effector T cells.

[0074] Cytotoxic T cells are a subgroup of CD8+ T cells that destroy infected cells.

[0075] Helper T cells are a subgroup of CD4+ T cells that mediate immune responses.

[0076] Finally, T cells having a chimeric antigen receptor (CAR) are also referred to as CAR-T cells and correspond to a specific cell engineering technology. These are T cells that express a chimeric antigen receptor. CAR-T cells can kill cancer cells by recognizing and binding to tumor antigens present on the cancer cells. The sample according to the present invention preferably comprises CAR-T cells.

[0077] The progenitor cells of the above immune cells may be mesenchymal stem cells.

[0078] The above cell sample may be derived from the patient subject to treatment through a biopsy or blood sample collection, in which case the patient and the donor are the same person. In this case, the obtained and preserved composition is administered to the same patient, and it is an autologous product.

[0079] Alternatively, the cell sample may be derived from other sources, such as from another person or using cell engineering, and in particular from a biopsy or blood sample collection. In this case, the obtained and preserved composition is administered to the patient subject to treatment rather than the donor: it is an allogenic product.

[0080] The sample may be packaged in a fresh state within a liquid medium, in which case the preservation period may be limited to a few hours, or it may be in the form of a frozen solution. The sample may contain cryopreservatives. The method according to the present invention preferably uses a fresh sample. The sample may be preserved with BSA (bovine serum albumin).

[0081] The above sample contains or may contain microorganisms. Microorganisms that can be isolated and / or detected according to the present invention may be bacteria, yeast, or fungi. The microorganisms may be aerobic or anaerobic. Examples of microorganisms that can be detected include Corynebacterium tuberculosteariticum, Moraxella catarrhalis, Moraxella osloensis, Bacteroides fragilis, Pichia carsonii, Bretanomyces bruxellensis, Paracoccus yeei, Saccharomyces cerevisiae, Streptococcus pyogenes, Penicillium expensum, Sphingomonas paucimobilis, Kocuria rhizophila, Staphylococcus Staphylococcus epidermidis, Aspergillus brasiliensis, Candida albicans, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Cutibacterium acnes, and Clostridium sporogenes There is.

[0082] To isolate microorganisms, the present invention proposes a method for isolating microorganisms directly from a sample. A sample of immune cells or their precursor cells comes into direct contact with a lysis composition. The sample of immune cells or their precursor cells does not undergo prior centrifugation, filtration, or selective agglutination processes to reduce the concentration of said cells.

[0083] This is because these preliminary steps can lead to microbial loss and result in false negatives. For example, high-speed centrifugation can promote adhesion between immune cells and certain bacteria, which may hinder their separation.

[0084] Accordingly, according to the present invention, it is necessary to perform selective lysis that destroys immune cells or their precursor cells in order to recover viable microorganisms. Accordingly, the addition of a chemical detergent or surfactant is commonly used to solubilize membrane proteins of mammalian cells. The detergent is an amphiphilic compound capable of forming micelles in an aqueous solution, with hydrophobic tails located inside the micelle and hydrophilic heads located outside the micelle. These micelles disrupt the phospholipid membrane. The detergent may be ionic, such as sodium dodecyl sulfate (SDS), milder nonionic, such as Triton X, or zwitterionic.

[0085] The cell wall of bacteria is rigid and is essentially composed of peptidoglycan. Teichoic acid is also present in Gram-positive bacteria (Gram+). This covalent bond network resists the dissolving action of surfactants for several minutes. Nevertheless, depending on the amount and type of surfactant, they can penetrate into the microorganism and destroy its protein structure, which can affect the microorganism's ability to proliferate and consequently affect its viability.

[0086] The complexity of selective lysis lies in selecting a lysis composition that effectively lyses immune cells and their progenitor cells while remaining non-toxic to microorganisms. In the present invention, it is essential for microorganisms to remain viable. Therefore, it is necessary to use a weak and non-toxic surfactant.

[0087] Furthermore, the efficiency of the lysis composition depends on the amount of cells present in the sample. The sample of the present invention consists of cells at a very high concentration. The efficiency also depends on the characteristics of the sample. A lysis composition effective against red blood cells is not necessarily effective against immune cells such as white blood cells. In fact, blood is mainly composed of red blood cells and contains about 4,000 to 7,000 white blood cells per 1 ml of blood. As small cells, red blood cells are lysed more easily than white blood cells.

[0088] Accordingly, according to the present invention, the sample is in contact with the following:

[0089] - A lysis composition comprising the following

[0090] o A lysis buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume

[0091] o and / or a lysis solution containing saponin at a concentration of 0.03% to 4% of the reaction volume

[0092] - Endonuclease capable of degrading nucleic acids released by the action of the above-mentioned dissolution composition

[0093] - Endopeptidase acting at pH 7 to 8.

[0094] The above-mentioned dissolving composition is used to destroy the cell membrane by acting on the physicochemical properties of the cell membrane.

[0095] The above-mentioned lysis composition preferably contains saponin. An example of saponin is Quillaja saponin. The cell membrane of leukocytes is more robust than the cell membrane of erythrocytes. In addition, immune cells or their precursor cells possess a nucleus and intracellular organelles, making them more resistant to lysis. Preferably, the final concentration of saponin is 0.03% to 2% of the reaction volume, more preferably 0.05% to 1%, even more preferably 0.05% to 0.8%, and even more preferably 0.05% to 0.2%.

[0096] The above-mentioned dissolution composition preferably comprises a nonionic detergent. The nonionic detergent is preferably a polyoxyethylene detergent. The detergent is preferably NP40. More preferably, the detergent is BRIJ. An example thereof is BrijO10. Preferably, the final concentration of the nonionic detergent is 0.004% to 0.050% of the reaction volume, more preferably 0.004% to 0.030% of the reaction volume.

[0097] In the absence of saponin, the final concentration of the nonionic detergent is 0.025% to 0.05% of the reaction volume.

[0098] In the absence of detergent, the final concentration of saponin is 0.10% to 4% of the reaction volume. Preferably, in the absence of detergent, the buffer contains a high concentration of salt, for example, a TRIZMA buffer exceeding 50 g / l.

[0099] Immune cells or their progenitor cells present at very high concentrations are partially dissolved by low concentrations of saponin and / or low concentrations of non-ionic detergent, whereas microorganisms present in very small amounts are not destroyed. Advantageously, in order not to damage microorganisms, the dissolving composition does not contain ionic detergents such as SDS.

[0100] The preparation of a dissolution composition comprising the preparation of a dissolution buffer containing a nonionic detergent is known to those skilled in the art. The components are dissolved and mixed.

[0101] In addition, the sample is contacted with an endopeptidase that acts effectively at pH 7 to 8.5; preferably, it acts at pH 7 to 8. The endopeptidase enables the complete lysis of immune cells. In fact, nonionic polyoxyethylene detergents, such as BRIJ and / or saponin, disrupt the plasma membrane of immune cells and their progenitor cells and increase permeability by forming complexes with lipids within the cell membrane. This mechanism subsequently causes the endopeptidase to hydrolyze membrane proteins, thereby rupturing the cells.

[0102] The endopeptidase used according to the present invention does not adversely affect the survival of microorganisms. Examples of endopeptidase include trypsin or endopeptidase mixtures such as Biowest’s Accutase® composition. The endopeptidase is preferably trypsin. Trypsin has optimal activity at pH 8. The trypsin may be in liquid form or in powder form that is reconstituted into a solution. The final concentration of trypsin is preferably in excess. For example, this may be greater than 0.1%.

[0103] According to the present invention, detection can be performed using a viability stain. Since immune cells or their progenitor cells can be labeled as microorganisms by the viability stain, complete lysis of immune cells can reduce false positives. Furthermore, since very small cell debris can be obtained by lysis, it is possible to prevent microorganisms from attaching to these debris and thereby prevent the microorganisms from being masked. Therefore, this method reduces false negatives.

[0104] In one embodiment, the sample is contacted with endopeptidase before contacting the soluble composition. This embodiment is particularly advantageous when the sample contains BSA.

[0105] In addition, pH must also be considered to optimize the efficiency of the lysis agents. Generally, the lysis agents have a pH of 5 to 9. Preferably, the lysis agents operate at a pH of 7 to 8.5, more preferably at a pH of 7 to 8.

[0106] Therefore, saponins and trypsin are very effective at basic pH values ​​of 7 to 8. In addition, at these pH values, the viability of target microorganisms is maintained, whereas at pH values ​​exceeding 8.5, it is not maintained in some target microorganisms.

[0107] After the addition of endopeptidase, a washing step with a suitable fluid is performed to stop enzymatic and chemical reactions. Preferably, Fluid D is used.

[0108] The above-mentioned lysis composition and endopeptidase are added to the sample in an appropriate amount and for a suitable time to lyse immune cells or their precursor cells while keeping potentially present microorganisms in a viable state.

[0109] Additionally, the sample is contacted with one or more enzymes capable of degrading nucleic acids. Such enzymes are known to those skilled in the art and are commonly used. The endonuclease non-specifically degrades all forms of DNA and RNA. An example is Benzonase® Nuclease commercially available from Merck Millipore. The final concentration of the endonuclease is preferably an excess. The final concentration may be 50 IU / ml to 100 IU / ml.

[0110] The above endonuclease may be separate from the soluble composition or may be part of the soluble composition. It may be added before, simultaneously with, or after the addition of the soluble composition and the endopeptidase. The above endopeptidase may be optionally inactivated before the endonuclease is added.

[0111] By degrading nucleic acids, the endonuclease prevents the filter from clogging. Consequently, filtration is improved, preventing false positives.

[0112] The above endonuclease may be in a dry form.

[0113] After the step of contacting the sample with the lysis composition and endopeptidase, the entire mixture is homogenized or mixed, for example, by vortexing or stirring. Additionally, an incubation step of several minutes accompanied by stirring may be performed.

[0114] Afterward, potentially surviving microorganisms are isolated from the sample by filtration. This isolation is performed on a porous membrane having pores smaller than the size of the bacteria.

[0115] Preferably, the porous membrane has pores of 0.30 to 0.50 μm, and more preferably, pores of 0.32 to 0.45 μm. Bacteria have a smaller diameter than yeast and fungi. Nevertheless, they are retained by the filter. Because the diameter of the membrane is very small, the dissolution of the sample must be very effective so that the filter does not clog and cause very large background fluorescence noise, which would obscure the specific fluorescence of the microorganisms. Thus, this method reduces false negatives.

[0116] The above microbial isolation step is a rapid step of less than 30 minutes, preferably less than 20 minutes.

[0117] After the microorganisms are isolated, they can be detected using any method, such as molecular biology or cytometry. Detection according to the present invention is performed using solid-phase cytometry, which enables the detection of potentially present viable microorganisms.

[0118] Accordingly, another aspect of the present invention is 10 6 to 10 8 The present invention relates to a method for detecting microorganisms potentially present in a sample containing immune cells or their precursor cells, said method comprising the following steps:

[0119] - Step of performing the separation method according to the present invention

[0120] - A step of labeling viable microorganisms retained on a porous membrane using a viability stain

[0121] - A step of scanning the porous membrane using a laser beam or a camera

[0122] - A step of determining the presence of surviving microorganisms captured in the above membrane.

[0123] Detection according to the present invention is particularly rapid, taking 4 to 6 hours, and detection of aerobic microorganisms is more rapid than detection of anaerobic microorganisms, which require longer incubation times. Solid-phase cytometry is a promising technique for sterility testing for ATMPs. This enables high-sensitivity detection of microorganisms. Accordingly, the SCANRDI® technology developed by the applicant enables the detection of any microbial presence in a filterable sample and provides results rapidly. Thus, the protocol according to the present invention enables ultra-fast sterility testing of samples of immune cells or their precursor cells within 6 hours. Such solid-phase cytometry enables the very rapid quantification of microorganisms (bacteria, yeast, or fungi) in a sample. The sample is filtered through a membrane, and all microorganisms present in the sample are recovered onto the membrane. Subsequently, the surviving microorganisms penetrate into the microorganism, are cleaved by esterase to release a fluorophore (fluorescein) into the microorganism, and are specifically labeled using a viability stain that causes the fluorophore to emit at a specific wavelength. Immune cells or their progenitor cells also contain esterase and can absorb the substrate in the same manner as the microorganisms. Therefore, labeling them with fluorescein may result in false positives. Thus, lysis of these cells is essential to distinguish the microorganisms present in the sample from immune cells or their progenitor cells. After the labeling step, a laser beam scans the entire surface of the membrane to excite the fluorophore, thereby enabling the detection and counting of any surviving microorganisms that may be present in the sample. A microscope coupled to the scanner allows for the visual confirmation of the microorganisms' presence.The present invention enables the achievement of a limit of detection of 1 CFU per sample.

[0124] In addition, one challenge in applying this technology to sterility testing for advanced therapeutic drugs is 10 5 cells / ml or more, preferably 10 6 Up to 5×10 7 It lies in the presence of a high cell concentration of cells / ml. In fact, such a non-filterable matrix on the membrane causes very large background fluorescence noise, which can mask the specific fluorescence of microorganisms present in very small amounts. Therefore, very effective lysis is required to obtain very small debris. The porous membrane of the solid-phase cytometer allows fluid and cell debris to pass through while retaining microorganisms. The membrane corresponds to a plurality of pores with an average diameter of less than 0.50 μm, preferably 0.32 to 0.45 μm.

[0125] Another aspect of the present invention relates to a lysis kit comprising the following:

[0126] - A lysis buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume

[0127] - A solution containing saponin at a concentration of 0.03% to 4% of the reaction volume

[0128] - Endonuclease

[0129] - Endopeptidase acting at pH 7 to 8.

[0130] In an alternative method, detection can be performed using molecular biology. In this case, this is 106 to 10 8 A method for detecting microorganisms potentially present in a sample containing immune cells or their precursor cells, said method comprising the following steps:

[0131] - Step of performing the separation method according to the present invention

[0132] - The step of culturing a sample in a nutrient medium that allows microorganisms to multiply.

[0133] - Step to remove or inactivate endonucleases

[0134] - Step of lysing microorganisms to recover nucleic acids

[0135] - A step of contacting the nucleic acid solution with primers so that the primers can amplify at least one gene or gene fragment of the microorganism

[0136] - A step of detecting the above microorganisms.

[0137] The isolation step of surviving microorganisms enables the removal of residual DNA.

[0138] The above endonuclease can be inactivated by diluting it by a large volume. This can be done before or after culture in a nutrient medium.

[0139] The present invention is described through the non-limiting embodiments presented below.

[0140] Examples

[0141] Example 1: Preparation of lysing agents

[0142] Dissolution buffer A was prepared with the following components:

[0143] - Trizma: 138 g

[0144] - H2O: 900 ml

[0145] The pH of the buffer solution was adjusted to 8.

[0146] Next, 4 μl of benzonase (Benzonase E1014-25KU, Sigma) was added to 7 ml of the previously prepared solution. 2 ml of trypsin (Trypsin EDTA: ref 25200-072-500 ml, Sigma) will be added during the dissolution protocol.

[0147] After preparing the following dissolution composition, 4 μl of benzonase (Benzonase E1014-25KU, Sigma) was added to 7 ml of the previously prepared solution. 2 ml of trypsin (Trypsin EDTA: ref 25200-072-500 ml, Sigma) will be added during the dissolution protocol.

[0148] Dissolved composition B comprises the following:

[0149] - Trizma (SIGMA / T6066-5KG standard): 138 g

[0150] - H2O: 900 ml

[0151] The pH of the buffer solution was adjusted to 8.

[0152] 1.5 g of saponin (Quillaja saponin, S4521-10G, Sigma) (1.5 g / l based on the soluble composition) was added to this buffer solution.

[0153] Dissolved composition C comprises the following:

[0154] - Trizma: 6 g

[0155] - BrijO10 (SIGMA / P6136-100G): 0.1296 g

[0156] - H2O: 900 ml

[0157] The pH of the buffer solution was adjusted to 8.

[0158] 1.5 g of saponin (Quillaja saponin, S4521-10G, Sigma) (1.5 g / l based on the soluble composition) was added to this buffer solution.

[0159] Dissolved composition D includes the following:

[0160] - Trizma: 6 g

[0161] - BrijO10: 0.1296 g

[0162] - H2O: 900 ml

[0163] - Sodium thiosulfate: Add 1 ml of stock solution (0.0499 g / l)

[0164] The pH of the buffer solution was adjusted to 8.

[0165] 1.5 g of saponin (Quillaja saponin, S4521-10G, Sigma) (1.5 g / l based on the soluble composition) was added to this buffer solution.

[0166] The dissolved composition E comprises the following:

[0167] - Trizma: 6 g

[0168] - NP40 (Nonidet P 40, Sigma, ref 74385-1L): 0.75 g

[0169] - H2O: 900 ml

[0170] The pH of the buffer solution was adjusted to 8.

[0171] 1.5 g of saponin (Quillaja saponin, S4521-10G, Sigma) (1.5 g / l based on the soluble composition) was added to this buffer solution.

[0172] The dissolved composition F includes the following.

[0173] - Trizma: 6 g

[0174] - NP40: 0.75 g

[0175] - H2O: 900 ml

[0176] The pH of the buffer solution was adjusted to 8.

[0177] The dissolved composition G includes the following:

[0178] - HEPES: Acidic HEPES 3.12 g, Basic HEPES 9.61 g

[0179] - BrijO10: 0.1296 g

[0180] - H2O: 900 ml

[0181] The pH of the buffer solution was adjusted to 8.

[0182] 1.5 g of saponin (Quillaja saponin, S4521-10G, Sigma) (1.5 g / l based on the soluble composition) was added to this buffer solution.

[0183] The dissolved composition H includes the following:

[0184] - HEPES: Acidic HEPES 3.12 g, Basic HEPES 9.61 g

[0185] - NP40: 0.75 g

[0186] - H2O: 900 ml

[0187] The pH of the buffer solution was adjusted to 8.

[0188] 1.5 g of saponin (Quillaja saponin, S4521-10G, Sigma) (1.5 g / l based on the soluble composition) was added to this buffer solution.

[0189] Example 2: Preparation of immune cell or progenitor cell samples

[0190] 1 ml of cells in RPMI 1640 medium (ref MS0A6Y100A Biowest) was brought into contact with 5 CFU (100 μl) of microorganisms.

[0191] The immune cells used are JURKAT cells or CAR-T cells:

[0192] - JURKAT cells (clone E6-1, ATCC TIB-152 or ECACC 88042803), 1 ml, 10 7 cells / ml

[0193] - CAR-T cells (ProMab PM-CAR2003-2M), 0.5 ml, 2×10⁻⁶ 6 cells / ml

[0194] - CAR-T cells (ProMab PM-CAR2003-2M), 1 ml, 4×10⁻⁶ 6 cells / ml

[0195] - T cells (patient-derived cells from the French Blood Bank (Etablissement francais du sang), 1 ml, 2×10⁶ 7 Cells / ml.

[0196] The above progenitor cells are as follows:

[0197] - Umbilical cord mesenchymal stem cells (patient-derived cells), 1 ml, 1×10⁶ 7 cell

[0198] - Lymphocytes, 1 ml, 2×10⁶ 7 cell

[0199] - Bone marrow-derived mesenchymal stem cells, 1.5 ml, 1.5×10⁶ 6 cell.

[0200] Example 3: Dissolution Protocol

[0201] The contaminated sample was brought into contact with 1 ml each of each dissolution composition according to Example 1 and benzonase.

[0202] This solution was incubated in an Eppendorf ThermoMixer C SN (37°C) at 37°C for 5 minutes under stirring conditions of 1000 rpm. After 5 minutes of incubation, 2 ml of trypsin was added, and the mixture was stirred again at 1000 rpm and 37°C for 5 minutes. Subsequently, the solution was diluted by adding several ml of Fluid D (ref 42624 BioMerieux) to stop the chemical and enzymatic reactions.

[0203] Example 4: Detection of microorganisms in dissolved samples

[0204] Afterward, this solution was filtered on a SCANRDI® device (membrane CB04 and ScanFilter 415701) through a membrane with a pore size of 0.32 to 0.38 μm. Fluid D can be used to perform multiple washes.

[0205] 1 ml of a CSE / CSM solution referred to as counterstains (CSE ref 205-R4070-01 bioMerieux / CSM 205-R4109-01, CSE diluted to a concentration of 1 / 100 in CSM) was added, maintained on a SCANRDI® membrane for 10 seconds, and then the CSE / CSM was passed through the membrane.

[0206] Afterward, the membrane was retrieved and placed on an activation pad, and an activation medium was applied to the pad (CELL BURST ACTIVATE kit bioMerieux ref 424512: CELL BURST ACTIVATE AER corresponds to TSB medium for aerobic microorganisms, and CELL BURST ACTIVATE ANAER corresponds to thioglycolate medium for anaerobic microorganisms). The pad was placed in an incubator at 30°C for 2 hours for aerobic microorganisms and under an anaerobic atmosphere (GEN bag Anaer bioMerieux 455534) for 2 hours and 30 minutes for anaerobic microorganisms.

[0207] Afterward, the pad was replaced, and a new pad containing the stain (ChemChrome V6 ref 201-R1007-03, diluted to 1 / 100 concentration in ChemSol B16 205-R2023-02) was used to incubate aerobic microorganisms for 45 minutes and anaerobic microorganisms for 1 hour and 30 minutes under an anaerobic atmosphere.

[0208] The stain used in SCANRDI® technology is a viability stain. SCANRDI® detects only viable microorganisms.

[0209] After the culture was completed, the membrane was recovered and analyzed using SCANRDI®.

[0210] Example 5: Cell lysis efficiency

[0211] Preparation of samples (Jurkat cells not containing microorganisms), lysis buffer A, and lysis protocols using lysis compositions B to H are described in Examples 1 to 4.

[0212] 1x10 7 Up to 1x10 8 Jurkat cells 1x10 7 Up to 1x10 8 Jurkat cells Dissolution buffer A Compositions B to H The JURKAT cells were lysed, but the excessive presence of fragments made it impossible to isolate the microorganisms. JURKAT: Efficient lysis of cells

[0213] SCANRDI readings show that the lysis protocol using buffer A results in the excessive presence of unfiltered debris. Therefore, this lysis protocol does not enable a method for isolating microorganisms from the sample. Conversely, lysis protocols using buffers B through H enable effective lysis of Jurkat cells, thereby allowing for the isolation of microorganisms.

[0214] Example 6: Cell lysis efficiency according to the concentrations of saponin, nonionic detergent, and endopeptidase

[0215] Sample preparation (microorganism-free JURKAT cells), lysis protocol, and readings by SCANRDI® are described in Examples 1, 2, 3, and 4.

[0216] JURKAT cell count 1x10 7 1.3x10 7 1.6x10 7 1.9x10 7 Volume of dissolution buffer D 1 ml 1 ml 1 ml 1 ml Total reaction volume 2 ml 2.3 ml 2.6 ml 2.9 ml Saponin % 0.075% 0.065% 0.0576% 0.0517% BRIJ O10 % 0.007% 0.006% 0.0049% 0.0044% 0.25% volume of trypsin 2 ml: 0.125% 2 ml: 0.115% 2 ml: 0.107% 2 ml: 0.10% SCANRDI readings Cell lysis Cell lysis Cell lysis Cell lysis

[0217] The results demonstrate that immune cells (JURKAT cells) can be effectively lysed. SCANRDI® readings on the membrane show that the lysis of cells and debris does not interfere with SCANRDI® readings for microbial counting.

[0218] Example 7: Results obtained for aerobic microorganisms by the detection method according to the present invention

[0219] The preparation of samples (Jurkat cells), the lysis protocol using lysis compositions B, D, and E, and detection are described in Examples 1, 2, 3, and 4. The microorganisms used are as follows: Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus pyogenes, Kocuria rhizophila Each microorganism was tested five times (five replicate experiments), and to verify the accuracy of the inoculation amount (5 CFU) on the dish, tests using five petri dishes were performed in parallel.

[0220] microorganism Dissolution buffer SCANRDI® results (average of 5 replicates) Petri dish results (average of 5 repeated experiments) Staphylococcus aureus D 3 3.6 Staphylococcus aureus B 66 57 Staphylococcus aureus E 77 64 Pseudomonas aeruginosa D 4 5.2 Pseudomonas aeruginosa B 49 45 Pseudomonas aeruginosa E 49 49 Bacillus subtilis D 4 6 Bacillus subtilis B 39 45 Bacillus subtilis E 46 41 Streptococcus pyogenes D 2 2 Kocuria rhizophila D 5.4 6.8

[0221] The results show that the microorganisms inoculated with the cells to undergo the lysis process remained viable and were detected by SCANRDI®. The obtained results indicate a sensitivity of less than 50 CFU for the tested microorganisms. All results satisfy the recommendations of pharmacopeias requiring a level of coverage of 50% to 200% between the Petri dish technique and the SCANRDI detection technique.

[0222] Example 8: Results obtained for anaerobic microorganisms by the detection method according to the present invention

[0223] Sample preparation (Jurkat cells), lysis protocols using lysis compositions B, D, and E, and detection for anaerobic microorganisms are described in Examples 1, 2, 3, and 4. The microorganisms used are as follows: Bacteroides fragilis, Clostridium sporogenes, Cutibacterium acnes .

[0224] microorganism Dissolution buffer SCANRDI® results (average of 5 replicates) Petri dish results (average of 5 repeated experiments) Bacteroides fragilis D 6 7 Clostridium sporogenes D 6 5.2 Cutibacterium acnes D 3 4.6 Cutibacterium acnes B 10 20 Cutibacterium acnes E 13 20

[0225] The results show that the anaerobic microorganisms inoculated with the cells to undergo the lysis process remained viable and were detected by SCANRDI®. The presence of thiosulfate in the detection protocol enabled a very high recovery rate.

[0226] The obtained results show a sensitivity of less than 30 CFU for the microorganisms tested.

[0227] Example 9: Anaerobic Microorganisms Candida albicans and Aspergillus brasiliensis The result obtained by the detection method according to the present invention with respect to

[0228] The preparation of samples (Jurkat cells), the lysis protocol using lysis compositions B, D, and E, and detection are described in Examples 1, 2, 3, and 4. The microorganisms used are as follows: Candida albicans, Aspergillus brasiliensis, Penicillium chrysogenum .

[0229] microorganism Dissolution buffer SCANRDI® results (average of 5 replicates) Petri dish results (average of 5 repeated experiments) Candida albicans D 3 4.6 Candida albicans B 27 49 Candida albicans E 26 50 Aspergillus brasiliensis D 3.4 4 Aspergillus brasiliensis B 47 37 Aspergillus brasiliensis E 43 38 Penicillium chrysogenum D 12 10

[0230] The results show that the anaerobic microorganisms inoculated with the cells to undergo the lysis process remained viable and were detected by SCANRDI®. The obtained results indicate a sensitivity of less than 50 CFU for the tested microorganisms.

[0231] Example 10: Effect of Saponin and Non-ionic Detergent Concentrations on Microbial Viability

[0232] According to Example 1 (Composition D), Example 2 (changing cell volume), and Example 3, the toxicity of the above protocol was tested using different microorganisms. The samples consist of JURKAT cells.

[0233] Following Example 3, in order to recover the microorganisms and grow them on petri dishes in a medium suitable for the type of microorganism, the solution was filtered on a NALGENE filter containing a membrane with a pore diameter of 0.45 μm.

[0234] Strain Cell volume Saponin % BRIJ % Filter recovery rate (%) relative to average inoculation Staphylococcus aureus (BB) 0.1 ml 0.1364% 0.0127% 126.26% 0.4 ml 0.1071% 0.0100% 126.26% 0.7 ml 0.0882% 0.0082% 142.42% 1 ml 0.075% 0.0070% 155.55% Pseudomonas aeruginosa (BB) 0.1 ml 0.1364% 0.0127% 100.53% 0.4 ml 0.1071% 0.0100% 95.14% 0.7 ml 0.0882% 0.0082% 89.06% 1 ml 0.075% 0.0070% 82.32% Bacillus subtilis (BB) 0.1 ml 0.1364% 0.0127% 80.74% 0.4 ml 0.1071% 0.0100% 80.74% 0.7 ml 0.0882% 0.0082% 83.33% 1 ml 0.075% 0.0070% 87.85% Aspergillus brasiliensis (BB) 0.1 ml 0.1364% 0.0127% 99.25% 0.4 ml 0.1071% 0.0100% 90.82% 0.7 ml 0.0882% 0.0082% 94.56% 1 ml 0.075% 0.0070% 87.07% Streptococcus pyogenes (BB) 0.1 ml 0.1364% 0.0127% 108.69% 0.4 ml 0.1071% 0.0100% 114.49% 0.7 ml 0.0882% 0.0082% 124.63% 1 ml 0.075% 0.0070% 121.73% Kocuria rhizophila (BB) 0.1 ml 0.1364% 0.0127% 100 0.4 ml 0.1071% 0.0100% 98.80% 0.7 ml 0.0882% 0.0082% 105.95% 1 ml 0.1500% 0.0140% 98.80% Clostridium sporogenes (BB550) 0.1 ml 0.1364% 0.0127% 80.49% 0.4 ml 0.1071% 0.0100% 90.32% 0.7 ml 0.0882% 0.0082% 70.66% 1 ml 0.075% 0.0070% 81.10% Bacteroides fragilis (prec) 0.1 ml 0.1364% 0.0127% 89.70% 0.4 ml 0.1071% 0.0100% 92.39% 0.7 ml 0.0882% 0.0082% 94.63% 1 ml 0.075% 0.0070% 84.34% Cutibacterium acnes (BB) 0.1 ml 0.1364% 0.0127% 102.40% 0.4 ml 0.1071% 0.0100% 96.38% 0.7 ml 0.0882% 0.0082% 94.37% 1 ml 0.075% 0.0070% 102.40% Moraxella catarrhalis (prec) 0.1 ml 0.1364% 0.0127% 89.34% 0.4 ml 0.1071% 0.0100% 72.16% 0.7 ml 0.0882% 0.0082% 81.32% 1 ml 0.075% 0.0070% 74.45% Sphingomonas paucimobilis (prec) 0.1 ml 0.1364% 0.0127% 92.59% 0.4 ml 0.1071% 0.0100% 86.20% 0.7 ml 0.0882% 0.0082% 98.97% 1 ml 0.075% 0.0070% 85.56% Escherichia coli (BB) 0.1 ml 0.1364% 0.0127% 103.99% 0.4 ml 0.1071% 0.0100% 99.17% 0.7 ml 0.0882% 0.0082% 99.17% 1 ml 0.075% 0.0070% 97.79% Staphylococcus epidermidis (BB30) 0.1 ml 0.1364% 0.0127% 105.12% 0.4 ml 0.1071% 0.0100% 102.56% 0.7 ml 0.0882% 0.0082% 101.28% 1 ml 0.075% 0.0070% 112.82% Streptococcus pneumoniae (prec) 0.1 ml 0.1364% 0.0127% 101.59% 0.4 ml 0.1071% 0.0100% 94.28% 0.7 ml 0.0882% 0.0082% 85.32% 1 ml 0.075% 0.0070% 94.28% Penicillium expansum 0.1 ml 0.1364% 0.0127% 65.36% 0.4 ml 0.1071% 0.0100% 61.47% 0.7 ml 0.0882% 0.0082% 74.45% 1 ml 0.075% 0.0070% 63.20%

[0235] The results obtained from the table above show that at a saponin concentration of 0.075 to 0.1364% of the reaction volume and a BRIJ concentration of 0.007 to 0.0127% of the reaction volume, T lymphocytes can be lysed without any toxicity to the microorganisms tested.

[0236] Example 11: Lysis of Jurkat cells (T lymphocytes) by a lysis composition and / or endopeptidase

[0237] 1) Tested Jurkat cell samples

[0238] BioMerieux Jurkat cell cultures harvested on September 15, 2022 were used, stored in 5% DMSO and 10% BSA, and thawed on September 16, 2022.

[0239] The above cells are diluted to 1 / 10 and 10 7 The concentration was adjusted to cells / ml (1 ml of cells was added to 9 ml of RPMI).

[0240] 2) Cell lysis

[0241] Lysis was performed according to Example 1. 1 ml of cells and 1 ml of a solution containing lysation composition C and endonuclease were added and treated for 5 minutes at 37°C while stirring at 1000 rpm (triple replicate experiment). After the lysation step, 200 μl of the lysates were collected in the first tube to perform measurements on a Gallios system (Beckman Coulter, 10-color Gallios).

[0242] 2 ml of 0.25% endopeptidase solution was added to the remaining tube. After incubating the tube at 37°C for 5 minutes, 200 μl of the lysate was collected to perform measurements on the Gallios system as before.

[0243] 3) Flow cytometry

[0244] The lysis effect on cells in step 2 was observed using a Gallios flow cytometer.

[0245] The table below shows the cytometric profiles of unlyzed cells, cells lysed using a lysis composition and endonuclease, and cells lysed using a lysis composition, endonuclease, and endopeptidase.

[0246] Untreated cells (%) Lysed cells (%) using lysis buffer C and endonuclease Cells lysed (%) using lysis buffer C, endonuclease, and endopeptidase % of surviving JURKAT cells 52.58 3.39 0.10 % of apoptotic cells 13.89 52.40 2.20 % of JURKAT cells in the form of cell fragments 31.49 39.52 96.72

[0247] Three groups of cells are observed:

[0248] - Survival Jurkat cell colony

[0249] - Apoptotic Jurkat cell colony

[0250] - Cell debris clusters

[0251] The lysis step (column 3) using the lysis composition and endonuclease induces loss of viability in Jurkat cells. Indeed, it is observed that virtually all cells have moved from the viable state to the dead cell window on a flow cytometer. A decrease in cell size is observed, which corresponds to the action of the lysis composition on the cell membrane. The saponins and BRIJs present in the lysis composition act by forming complexes with membrane lipids, thereby structurally disrupting the cell's plasma membrane and increasing its permeability.

[0252] This mechanism subsequently causes endopeptidase to hydrolyze membrane proteins, thereby rupturing the cells (Column 4). Consequently, it is observed that virtually all apoptotic cells have moved from the apoptotic cell region to the cell debris window. The action of trypsin also continues, breaking down the nuclear membrane and releasing intracellular substances.

[0253] In conclusion, the above-mentioned lysis composition and endopeptidase act synergistically to enhance the lysis of immune cells.

Claims

Claim 1 10 6 to 10 8 A method for isolating viable microorganisms potentially present in a sample containing immune cells or their precursor cells, comprising the following steps: - bringing the sample into contact with the following: A dissolution composition comprising the following o A dissolution buffer comprising a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume, and / or o A dissolution solution containing saponin at a concentration of 0.03% to 4% of the reaction volume; An endonuclease capable of digesting nucleic acids released by the action of the above-mentioned dissolution composition; Endopeptidase acting at pH 7 to 8 - a step of isolating potentially viable microorganisms present in the sample by filtering the sample through a filter having a pore diameter of 0.30 to 0.50 μm. Claim 2 A method according to claim 1, characterized in that the sample is in contact with the following: A dissolution composition comprising a dissolution buffer containing a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume and a saponin at a concentration of 0.03% to 4% of the reaction volume; An endonuclease capable of degrading nucleic acids released by the action of the above-mentioned dissolution composition; Endopeptidase acting at pH 7 to 8. Claim 3 In claim 1 or 2, the sample is 10 6 Up to 5×10 7 It comprises immune cells or their precursor cells, preferably 5×10 6 to 10 7 A method characterized by including a number of cells. Claim 4 A method according to any one of claims 1 to 3, characterized in that the immune cell is a CAR-T cell. Claim 5 A method characterized in that, in any one of claims 1 to 4, the endopeptidase is trypsin. Claim 6 A method characterized in that, in any one of claims 1 to 5, the nonionic detergent is a polyoxyethylene detergent. Claim 7 A method according to claim 5, characterized in that the polyoxyethylene detergent is NP40. Claim 8 A method according to claim 5, characterized in that the polyoxyethylene detergent is BRIJ. Claim 9 A method characterized in that, in any one of claims 1 and 3 to 8, when saponin is absent, the final concentration of the nonionic detergent is 0.025% to 0.05%. Claim 10 A method characterized in that, in any one of claims 1 and 3 to 8, when no nonionic detergent is present, the final concentration of saponin is 0.10% to 4% of the reaction volume. Claim 11 A method according to any one of claims 1 to 10, characterized in that the final concentration of the endonuclease is greater than 0.1% of the reaction volume. Claim 12 A method according to any one of claims 1 to 11, wherein the endonuclease is in contact with the sample before the endopeptidase, or is in contact with the sample after the inactivation of the endopeptidase. Claim 13 A method characterized in that, in any one of claims 1 to 12, the final concentration of the endopeptidase is an excess. Claim 14 A method according to any one of claims 1 to 13, wherein the porous membrane has pores of 0.32 to 0.45 μm. Claim 15 A method characterized in that, in any one of claims 1 to 14, the sample is a fresh sample. Claim 16 10 6 to 10 8 A method for detecting microorganisms potentially present in a sample containing immune cells or their precursor cells, comprising the following steps: - performing a separation method according to any one of claims 1 to 15; - labeling viable microorganisms retained on a porous membrane using a viability stain; - scanning the porous membrane using a laser beam or a camera; - determining whether viable microorganisms captured by the membrane are present. Claim 17 A dissolution kit comprising: - a dissolution buffer comprising a nonionic detergent at a concentration of 0.004% to 0.050% of the reaction volume; - a solution comprising saponin at a concentration of 0.03% to 4% of the reaction volume; - an endonuclease; - an endopeptidase acting at pH 7 to 8.

Images