Method for Recovering Cells from a Urine Sample Using Hyaluronidase

Abstract

The present invention relates to a method for recovering cells from a urine sample using hyaluronidase. More specifically, in order to reduce the loss of cells in the urine, the present invention may include the steps of centrifuging a urine sample to obtain a precipitate containing cells, performing hyaluronidase treatment on the precipitate to dissociate or disperse aggregates, washing the precipitate, and then filtering it using a filter having a predetermined pore size. According to the present invention, it is possible to reduce cell loss throughout the process and improve the cell yield, and also provide the effect of improving the purity of the obtained cell population and the proliferation efficiency during culture. Accordingly, urine can be utilized as a stable source of cells.

Description

Technology Field

[0001] The present invention relates to a method for recovering cells from a urine sample using hyaluronidase. More specifically, in order to reduce the loss of cells in the urine, the present invention may include the steps of centrifuging a urine sample to obtain a precipitate containing cells, performing hyaluronidase treatment on the precipitate to dissociate or disperse aggregates, washing the precipitate, and then filtering it using a filter having a predetermined pore size.

[0002] According to the present invention, it is possible to reduce cell loss throughout the process and improve the cell yield, and also provide the effect of improving the purity of the obtained cell population and the proliferation efficiency during culture. Accordingly, urine can be utilized as a stable source of cells. Background Technology

[0004] Urine has the advantages of being non-invasively collected from the human body, allowing for repeated sample collection, and placing a significantly lower burden on the subject compared to blood sampling. Furthermore, it is known that urine contains various cells, including urothelial cells, kidney-derived cells, immune cells, and stem cell-like cells.

[0005] In particular, urine-derived stem cells are attracting attention as a useful cell source in various fields, such as regenerative medicine, tissue engineering, disease modeling, drug screening, and cell therapy development, as they have been reported to exhibit high proliferative capacity and differentiation potential while being obtainable through relatively simple methods.

[0006] However, despite these advantages, there are still limitations in reliably obtaining a sufficient number of cells from urine. In conventional technology, physicochemical environmental factors such as urine pH, osmotic pressure, and salt concentration, proteolytic enzymes and toxic components, and cell death during storage or transport have been identified as major causes; these have primarily been recognized as issues related to reduced cell viability or decreased culture efficiency.

[0007] Furthermore, conventional cell separation and recovery processes have not sufficiently considered structural elements present in urine, particularly aggregated structures containing cells. Generally, cells in urine samples are recovered through filtration and centrifugation, but these processes have not adequately reflected the physical distribution of the cells.

[0008] However, according to repeated observations by the inventors, mucus-like aggregates (or mucus-like masses) in which mucinous substances and cells are aggregated together exist within urine samples, and these structures are observed in the form of translucent gels or clumps. Furthermore, it was confirmed that said mucus-like aggregates possess physical stability that prevents them from easily dissociating through simple pipetting or conventional cell separation processes.

[0009] In particular, microscopic observation revealed that the interior or surface of the mucous aggregate contained numerous urine-derived cells, and that some cells were found to be trapped inside the aggregate or attached to the surface.

[0010] However, in conventional technology, cell separation processes including filtration have been performed without sufficiently considering the presence of such mucous aggregates; consequently, a problem arose where the cells contained within the aggregates were lost along with the aggregates as they were removed by the filter. As a result, there was an inherent limitation in that the number of cells recoverable from urine was restricted.

[0011] Therefore, conventional cell recovery methods that do not consider the structural characteristics of urine samples have limitations in terms of cell acquisition efficiency and reproducibility, and face difficulties in application in fields such as single-cell analysis, flow cytometry, and cell therapy manufacturing, where securing high-purity and high-yield cells is required. The problem to be solved

[0013] The present invention aims to provide a cell recovery method that minimizes cell loss occurring during the process of obtaining cells from a urine sample, maintains cell viability, and improves the cell yield through a pretreatment step.

[0014] Furthermore, the present invention aims to effectively dissociate or disperse cells contained in mucinous aggregates within urine samples to prevent cell loss occurring during the filtration process and to improve cell recovery efficiency. It also aims to provide a pretreatment process capable of effectively degrading mucinous aggregates while maintaining cell viability by optimizing enzyme treatment conditions, and at the same time, a cell recovery method capable of effectively removing impurities while simultaneously improving the recovery rate and culture efficiency of target cells by performing the pretreatment step and the filtration step sequentially.

[0016] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0018] The present invention aims to provide a method for recovering cells from a urine sample using hyaluronidase.

[0020] According to one embodiment, the present invention

[0021] (a) A step of centrifuging a urine sample;

[0022] (b) a step of treating the centrifuged sample with hyaluronidase;

[0023] (c) a step of washing the sample treated with the above hyaluronidase; and

[0024] (d) A method for recovering cells is provided, comprising the step of separating cells by filtering the sample with a filter.

[0025] In the present invention, the centrifugation may be performed at 400 × g to 600 × g for 5 minutes to 15 minutes.

[0026] In the present invention, the hyaluronidase can be treated at a concentration of 10 μg / mL to 1000 μg / mL.

[0027] In the present invention, the hyaluronidase treatment may be performed for 5 to 20 minutes.

[0028] In the present invention, the hyaluronidase treatment can be performed at 35°C to 40°C.

[0029] In the present invention, the washing may be performed one or more times using an isotonic buffer solution or a culture medium.

[0030] In the present invention, the filter may have a pore size of 10 μm to 40 μm.

[0031] In the present invention, the filter may have a pore size of 20 μm.

[0033] According to one embodiment of the present invention, mucous aggregates in a urine sample are effectively dissociated or dispersed so that cells are homogeneously dispersed in the form of single cells, thereby reducing cell loss during the filtration process and improving the overall cell yield. In addition, the recovered cells maintain high viability and colonization ability, so they can be usefully utilized in subsequent culture and cell analysis processes. Effects of the invention

[0035] According to the present invention, cells can be homogeneously dispersed in a single-cell form by effectively dissociating or dispersing mucous aggregates present in a urine sample, and accordingly, the phenomenon of cells being removed along with aggregates during cell separation, washing, and filtration processes can be significantly reduced, thereby improving the overall cell yield.

[0036] In addition, the pretreatment process according to the present invention can stably maintain or improve cell viability while minimizing physical stress applied to cells, and the recovered cells can improve cell proliferation ability in subsequent culture processes by containing a large amount of cells with excellent colony-forming unit (CFU) capacity.

[0037] In addition, the present invention can selectively remove non-purpose cells such as squamous epithelium or residual aggregates by applying a filtration process using a filter having a predetermined pore size, thereby securing a more homogeneous cell population.

[0038] Furthermore, the pretreatment and separation process according to the present invention can significantly reduce non-specific binding and impurity incorporation that may occur during the magnetic-activated cell sorting (MACS) process by effectively removing impurities from the sample in advance, and accordingly, can improve the separation efficiency and purity of target cells, such as an increase in the proportion of CD326 (EpCAM) positive cells.

[0039] In addition, the cell recovery method according to the present invention can improve the efficiency of securing urine-derived cells and provide a stable cell source that can be utilized in various fields such as single-cell analysis, flow cytometry, biomarker discovery, cell culture, and cell therapy research. Brief explanation of the drawing

[0041] Figure 1 illustrates the process of separating cells from a urine sample. Figure 2 shows the results of staining mucous aggregates in a urine sample with Alcian blue. Figure 3 shows the results of comparing a conventional process (control group) and a hyaluronidase-treated group by staining them with Alcian blue. Figure 4 shows a comparison of cell viability after treatment with hyaluronidase at different concentrations. Figure 5 shows the results of observing changes in mucinous aggregates after treatment with hyaluronidase at different concentrations through Alcian blue staining. Figure 6 shows a comparison of the number of surviving cells according to the conventional process and the process of the present invention. Figure 7 shows the results of comparing the number of colonies formed at the end of culture after culturing cells recovered from a conventional process and the process of the present invention. Figure 8 shows the number of surviving cells at the end of culture after culturing cells recovered from a conventional process and the process of the present invention. Figure 9 shows the results of comparing the expression of CD326 (EpCAM) and Desmoglein-3 (DSG-3) by performing self-activated cell separation (MACS) and flow cytometry (FACS) on cell populations recovered according to the conventional process and the process of the present invention. Figure 10 shows the degree of dissociation or dispersion of mucinous aggregates in a conventional process, a DNase-treated group, a hyaluronidase-treated group, and a group treated with both DNase and hyaluronidase, compared through Alcian blue staining. Figure 11 shows a comparison of cell survival rates according to each treatment group. Figure 12 shows a comparison of the number of surviving cells recovered from the conventional process and each enzyme treatment group. Figure 13 shows a comparison of the number of colonies formed at the end of culture after culturing cells recovered from the conventional process and each enzyme treatment group. Figure 14 shows a comparison of the total number of surviving cells at the end of culture after culturing cells recovered from the conventional process and each enzyme treatment group. Specific details for implementing the invention

[0042] The present invention will be described in detail below. However, this is presented as an example of the invention and does not limit the scope of the invention, and it is obvious to those skilled in the art that various modifications to the embodiments are possible within the scope of the invention. Throughout this specification, unless otherwise specifically stated, "includes" or "contains" refers to the inclusion of any component (or constituent) without any particular limitation and should not be interpreted as excluding the addition of other components (or constituents).

[0044] The present invention relates to a pretreatment process for efficiently recovering cells from a urine sample and a method for recovering cells including the same, and is intended to minimize cell loss in the urine sample and improve recovery efficiency and purity.

[0045] The above urine sample may be derived from mammals, for example, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but is not limited thereto.

[0046] In particular, the present invention relates to a method for effectively recovering cells through a series of processes including centrifugation, dissociation or dispersion of mucous aggregates, washing, and filtration using a filter having a specific pore size.

[0047] In this specification, “pretreatment” means a step of treating a urine sample or a centrifuged sample with hyaluronidase to dissociate or disperse mucous aggregates.

[0049] The cell recovery method according to the present invention is,

[0050] (a) A step of centrifuging a urine sample;

[0051] (b) a step of treating the centrifuged sample with hyaluronidase;

[0052] (c) a step of washing the sample treated with the above hyaluronidase; and

[0053] (d) The method may include a step of separating cells by filtering the sample with a filter.

[0055] Hereinafter, each step according to the present invention will be described in more detail.

[0057] First, the collected urine sample is centrifuged.

[0058] The above centrifugation can be performed to effectively concentrate cells while minimizing cell damage.

[0059] In one embodiment, the centrifugation may be performed for about 5 minutes to 15 minutes under conditions of about 400 × g to 600 × g.

[0060] According to one exemplary embodiment, it was confirmed that when centrifugation is performed under the above conditions, the cell recovery rate is stably secured and cell damage caused by excessive centrifugal force is suppressed.

[0061] Through such a centrifugation process, a sample containing cells and mucinous aggregates can be formed.

[0063] Next, the step of treating the centrifuged sample with hyaluronidase is performed.

[0064] The above hyaluronidase plays a role in dissociating or dispersing aggregates by breaking down extracellular matrix components constituting mucinous aggregates, particularly hyaluronic acid.

[0065] In one embodiment, the hyaluronidase may be treated at a concentration of about 10 μg / mL to 1000 μg / mL and reacted at a temperature of about 35°C to 40°C for about 5 to 20 minutes.

[0066] According to one exemplary embodiment, it was confirmed that through the hyaluronidase treatment, mucous aggregates in urine are effectively dissociated and cells are homogeneously dispersed in the form of single cells, and consequently, the phenomenon of cells being removed along with aggregates during filtration and washing processes is significantly reduced, thereby increasing the total cell yield. In addition, it was confirmed that while a decrease in cell viability was observed under high concentration conditions, cell viability was stably maintained within an appropriate concentration range.

[0068] Next, a step of washing the sample treated with the hyaluronidase is performed.

[0069] The above washing step is intended to stop the enzyme reaction and remove residual enzymes and impurities, and can be performed using a culture medium or a buffer solution.

[0070] In one embodiment, the washing may be performed one or more times using an isotonic buffer solution or a culture medium, and centrifugation may be performed as needed.

[0071] According to one exemplary embodiment, it was confirmed that by performing the washing step, additional cell damage caused by residual enzymes is prevented, and the recovered cells contain a large amount of cells with excellent colony-forming unit (CFU) capacity, thereby improving cell proliferation ability in the subsequent culture process.

[0073] Next, the step of separating cells by filtering the above sample is performed.

[0074] The above filtration is intended to remove relatively large cells or residual aggregates based on differences in cell size, and to selectively recover cells dispersed in the form of single cells.

[0075] In one embodiment, the filter may have a pore size of about 10 μm to 40 μm, and preferably, a filter having a pore size of about 20 μm may be used.

[0076] According to one exemplary embodiment, when a filter having a pore size of about 20 μm is applied, non-purpose cells such as squamous epithelium and residual aggregates are effectively removed, while purpose cells dispersed in the form of single cells pass through the filtration, and it was confirmed that the purity of the cell population is improved while the overall cell recovery rate is maintained or increased.

[0078] In particular, in the present invention, the dissociation or dispersion process of the mucous aggregate is performed prior to the filtration step, thereby converting the cells contained within the aggregate into a single-cell form so that they can smoothly pass through the filtration process.

[0079] According to one exemplary embodiment, when the above-described process sequence was applied, it was confirmed that cell loss due to aggregates was significantly reduced compared to the conventional process, the total cell yield increased, and the recovered cells exhibited high CFU formation ability. Furthermore, it was confirmed that when the magnetic-activated cell sorting (MACS) process was applied, non-specific binding and impurity incorporation were reduced, thereby improving the purity of CD326 (EpCAM) positive cells.

[0081] After filtration, additional washing and centrifugation steps can be performed as needed to further purify the cells.

[0082] The finally recovered cells can be resuspended in a culture medium for cultivation or used in cell analysis or separation processes, and can be utilized as a high-quality cell source applicable to various fields such as single-cell analysis, biomarker discovery, and cell therapy research.

[0084] In addition, the cells obtained as described above can be utilized in combination with various cell separation technologies, such as self-activating cell separation technology.

[0086] The present invention will be described in detail below through examples, but the following examples and experimental examples are merely illustrative of one form of the present invention and the scope of the present invention is not limited by the following examples and experimental examples.

[0088] <Example>

[0089] Example 1. Confirmation of the presence and dissociation or dispersion of mucinous aggregates in urine via Alcian blue staining

[0090] In this embodiment, the presence of mucous aggregates in a urine sample and whether the aggregates dissociated or dispersed were confirmed.

[0091] After collecting urine samples, centrifugation was performed at a rate of approximately 400 × g to 600 × g for about 15 minutes to recover the lower layer containing pellets and mucinous aggregates. A portion of the recovered lower layer was taken, stained with Alcian blue staining solution, and observed under a microscope. As a result, the structure of mucinous aggregates stained by Alcian blue was observed, and a form containing cells inside or around the aggregates was confirmed (Fig. 2). In other words, the presence of mucinous aggregates in the urine samples was confirmed.

[0092] Meanwhile, to dissociate or disperse the mucinous aggregates, hyaluronidase was dissolved in isotonic buffer (HBSS, pH approx. 7.2) to prepare a stock solution. The stock solution was stored at approximately -20°C and diluted immediately before use. Approximately 0.5 mL to 1.0 mL of the diluted hyaluronidase solution was added to the recovered subsoil, and the solution was resuspended uniformly by light pipetting. Subsequently, the reaction was carried out at approximately 37°C for 5 to 15 minutes. For comparison, a conventional process group (control group) with PBS added without enzyme treatment was prepared under the same conditions. After the reaction was completed, culture medium or buffer solution was added to neutralize the enzyme reaction, and then the cells were recovered by re-centrifugation at approximately 400 × g to 600 × g. Subsequently, Alcian Blue staining was performed again for observation.

[0093] As a result, it was confirmed that while the mucilaginous aggregate structure was maintained in the conventional process group (control group), the mucilaginous aggregate structure was reduced or observed in a dispersed form in the hyaluronidase-treated group (Fig. 3). In other words, it was confirmed that the dissociation or dispersion of mucilaginous aggregates was induced by hyaluronidase treatment.

[0095] Example 2. Evaluation of mucinous aggregate dissociation effect and cell viability according to hyaluronidase concentration

[0096] In this example, the dissociation effect of mucinous aggregates and cell viability were evaluated according to hyaluronidase concentration.

[0097] Samples were prepared in the same manner as in Example 1, but experimental groups were formed by applying hyaluronidase at various concentrations. Each experimental group included a conventional process group (control group). After performing enzyme treatment under the same conditions for each experimental group, cells were recovered, and cell viability was evaluated by distinguishing between live and dead cells using trypan blue staining.

[0098] As a result, cell viability was maintained at a level similar to that of the conventional process group within a certain concentration range, and a trend of increasing viability was observed under some conditions. On the other hand, a trend of decreasing cell viability was confirmed under high concentration conditions of 5000 μg / ml (Fig. 4). In other words, it was confirmed that cell viability is stably maintained within a specific concentration range.

[0099] In addition, the degree of dissociation of mucinous aggregates according to hyaluronidase concentration was observed through Alcian blue staining, and it was confirmed that the dissociation or dispersion effect tended to increase with increasing concentration, while aggregates remained at a low concentration of 10 μg / ml (Fig. 5). In other words, an enzyme concentration-dependent dissociation effect was confirmed.

[0101] Example 3. Evaluation of increased cell recovery yield and culture characteristics following hyaluronidase treatment

[0102] In this example, the cell recovery rate and culture characteristics were evaluated when the mucous aggregate dissociation or dispersion process was performed prior to the filtration process.

[0103] The conventional process group recovered cells by passing urine samples, which had not undergone separate enzyme pretreatment, directly through a filter (cell strainer) with a pore size of approximately 20 μm, while the process group of the present invention recovered cells using the same filter after performing 250 μg / mL hyaluronidase pretreatment. The number of viable cells recovered from each group was counted using trypan blue staining.

[0104] As a result, it was confirmed that the amount of recovered cells in the process group of the present invention increased significantly compared to the conventional process group (Fig. 6). In addition, when the recovered cells were cultured for 14 days, the number of colonies formed (Fig. 7) and the total number of surviving cells at the same time point (Fig. 8) were also found to increase significantly compared to the conventional process group (control group).

[0106] Example 4. Characterization of obtained cell population by cell surface marker analysis

[0107] In this embodiment, the characteristics of the cell population obtained through the process of the present invention were evaluated.

[0108] The cells recovered in Example 2 were separated using self-activated cell separation (MACS) targeting CD326 (EpCAM), and then flow cytometry (FACS) was performed to analyze the expression of CD326 (EpCAM) and DSG-3.

[0109] As a result, when the process of the present invention was applied, it was confirmed that the proportion of CD326 (EpCAM) positive cells increased compared to the conventional process group (control group) (gating area P3 in Fig. 9), and the proportion of DSG-3 positive cells decreased (gating area P4 in Fig. 9). In other words, it was proven that non-target cell incorporation was reduced and the purity of the target cell population was improved.

[0111] Example 5. Quantitative Comparative Evaluation of 4 Groups According to Mucinous Aggregate Dissociation Method

[0112] This example quantitatively compared the dissociation effect of mucinous aggregates and cell characteristics according to the conventional process, DNase treatment, hyaluronidase treatment, and combined treatment of DNase and hyaluronidase.

[0113] 5-1. Experimental Method

[0114] The sample preparation, enzyme treatment, cell recovery, and culture conditions of this example were performed in the same manner as in Examples 1 and 2. Each group was composed as follows.

[0115] - Conventional process group (control group, enzyme untreated)

[0116] - DNase-treated group

[0117] - hyaluronidase-treated group

[0118] - DNase + hyaluronidase combination treatment group

[0119] Subsequent evaluations were performed using the same method as in Examples 2 to 4.

[0120] 5-2. Results

[0121] (1) Comparison of the degree of dissociation of mucinous aggregates (Fig. 10)

[0122] Alcian blue staining was performed on each group to compare the degree of dissociation of mucinous aggregates (Fig. 10). In the conventional process group, the structure of the mucinous aggregates was maintained, whereas in all enzyme-treated groups, the aggregates were observed to be reduced or dispersed.

[0123] (2) Cell viability (Fig. 11)

[0124] The hyaluronidase-treated group maintained a cell viability similar to that of the conventional treatment group, whereas the DNase-treated group and the combined treatment group showed a tendency for cell viability to decrease.

[0125] (3) Total number of surviving cells (Fig. 12)

[0126] In the hyaluronidase-treated group, a trend of increasing survival cell counts was observed compared to the conventional treatment group, while the DNase-treated group showed a decreasing trend, and the combination treatment group showed a level similar to the conventional treatment group.

[0127] (4) Number of colonies formed (Fig. 13)

[0128] The number of colonies formed was highest in the hyaluronidase-treated group, the DNase-treated group showed a decreasing trend, and the combined treatment group showed a level similar to the conventional treatment group.

[0129] (5) Total number of surviving cells after culture (Fig. 14)

[0130] The total number of surviving cells at the end of culture also showed the same trend as above.

[0131] Therefore, although mucinous aggregate dissociation can be induced by both DNase and hyaluronidase treatments, it was confirmed that hyaluronidase treatment alone showed the best effect in terms of cell viability and recovery efficiency.

[0133] Specific parts of the present invention have been described in detail above. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A method for recovering cells from a urine sample, comprising: (a) centrifuging the urine sample; (b) treating the centrifuged sample with hyaluronidase to dissociate or disperse mucous aggregates present in the centrifuged sample; (c) washing the sample treated with hyaluronidase; and (d) filtering the sample with a filter having a pore size of 10 μm to 40 μm to separate the cells. Claim 2 A method according to claim 1, wherein the centrifugation is performed at 400 × g to 600 × g for 5 to 15 minutes. Claim 3 A method according to claim 1, wherein the hyaluronidase is treated at a concentration of 10 μg / mL to 1000 μg / mL. Claim 4 A method according to claim 1, wherein the hyaluronidase treatment is performed for 5 to 20 minutes. Claim 5 In claim 1, the method wherein the hyaluronidase treatment is performed at 35°C to 40°C. Claim 6 A method according to claim 1, wherein the washing is performed one or more times using an isotonic buffer solution or a culture medium. Claim 7 delete Claim 8 In claim 1, the filter has a pore size of 20 μm.

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