CN112843256A

CN112843256A - Cell capable of tracing in organism, preparation method thereof and application thereof in cell pharmacokinetics research

Info

Publication number: CN112843256A
Application number: CN201911173634.6A
Authority: CN
Inventors: 彭会歌; 韩立; 潘国宇
Original assignee: Shanghai Weizhizhuo Biological Technology Co ltd
Current assignee: Shanghai Weizhizhuo Biological Technology Co ltd
Priority date: 2019-11-26
Filing date: 2019-11-26
Publication date: 2021-05-28
Also published as: WO2021103288A1

Abstract

The invention discloses a cell capable of being traced in a living body, a preparation method thereof and application thereof in cell pharmacokinetics research, relating to the technical field of biological medicines. The invention discloses a cell modification method and also discloses a cell which can be traced in an organism, wherein the cell is marked with a fluorescent dye, and the fluorescent dye is coupled through a chemical bond and is combined on the surface of the cell in an irreversible mode. The cell is directly marked with the fluorescent dye, the marking of the fluorescent dye has no influence on cell proliferation, the cell medicine has better stability, can be traced in vivo, accurately and quickly reflects the change process or distribution condition of the cell in vivo, and can be applied to cell pharmacokinetic research.

Description

Cell capable of tracing in organism, preparation method thereof and application thereof in cell pharmacokinetics research

Technical Field

The invention relates to the technical field of biomedicine, in particular to a cell which can be traced in an organism, a preparation method thereof and application thereof in cell pharmacokinetics research.

Background

Cell medicine has become one of the latest fields in medicine research and development, and a large number of clinical research results show that cell therapy has good curative effect in the fields of cancer, arthritis, central nervous system lesion and the like. No matter what form of the medicine, the efficacy and toxicity of the medicine coexist, which is the theoretical basis for the research of the pharmacokinetics, efficacy and toxicity (PK, PD, TD) of the medicine, so that the research of the change of the cell therapy medicine in vivo is very critical for judging the curative effect and the possible toxic and side effect. However, there is a lack of understanding of the in vivo processes of cellular drugs relative to the relatively mature in vivo pharmacokinetic studies of small and large molecular drugs. Taking the CAR-T product KYMRIAH approved by nova as an example, the examiners unfortunately indicate while confirming the therapeutic effect: if systemic studies can be carried out to find the appropriate dose, this may not only improve the therapeutic effect but also reduce the cytokine storm.

Based on this, it is necessary to systematically study the pharmacokinetic properties of cellular drugs by using the conventional pharmacokinetic theory and combining the characteristics of cellular drugs. To date, no international relationship has been established between the in vivo potency of cellular drugs and the pharmacokinetics of cellular drugs in animals or humans. This is because the pharmacokinetics of cellular drugs, whether by detection methods or kinetic theory, are very different from classical pharmacokinetic studies of the past. For example, the in vivo clearance of small molecule drugs often has first order kinetic characteristics, the in vivo properties of which can be fitted and predicted by mathematical models. Cellular drugs have proliferative capacity and often have non-linear characteristics in their concentration change after entry into the body.

The problem to be solved for examining the in vivo distribution of cell drugs is the in vivo detection method of cells, and the current in vivo detection means of cells can be mainly divided into two categories, namely invasive (collecting body fluid and measuring mRNA, specific protein and the like of cells by tissue qPCR method) and non-invasive (in vivo imaging technology and the like). The qPCR method uses a human-derived special gene fragment as a biomarker by means of species difference, realizes quantitative analysis of human-derived cells in animal blood and tissue samples by detecting the content of human-derived DNA, detects the distribution and removal of the human-derived cells in animal bodies, can semi-quantitatively or even quantitatively determine the number of cells in a specific tissue, and has high accuracy. Although the qPCR method is used only within the limits (between species and sex), the operation is cumbersome, and the detection limit is low, due to its relatively high accuracy, the qPCR method is mainly used for the current study of the in vivo time course and pharmacokinetic properties of the therapeutic cells. The present inventors hoped to find other simpler quantification methods that can replace the qPCR quantification method by non-invasive labeling methods for pharmacokinetic studies of cellular drugs. The existing small animal living body imaging technology, isotope labeling and the like can qualitatively reflect the in vivo distribution of cell medicines on the whole level, cells are labeled in vitro in a non-specific way by means of fluorescence, transgenosis, isotopes or magnetic ions and the like, and then the distribution and the clearing condition of the labeled cells are judged by detecting labeled signals in vivo. However, the labeling means may affect the activity of the cells, for example, isotopic labeling may impair the viability and differentiation ability of the treated cells, compared to a method of labeling with a dye, which is more safe. However, in the dye-labeling method, endocytosis dye such as DiR is used for labeling cells, so that the efficiency is high, but the labeling has no specificity, and after the labeled cells enter the body and are metabolized, DiR may enter autologous tissue cells, so that the in vivo process of the labeled cells cannot be accurately reflected. In addition, in comparison with the disclosed cell fluorescent labeling method (patent No. ZL201610237612.1), it is necessary to select an appropriate antibody molecule according to the target cell, label and label the target cell with the fluorescently labeled antibody molecule, and once the target cell is changed, the type of the antibody needs to be changed, which is a complicated procedure.

In summary, there is an urgent need in the art to further develop a convenient and efficient in vivo cell detection method to meet the requirements of cell pharmacokinetics research.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a cell which can be traced in an organism, a preparation method and a cell modification method thereof. The cell which can be traced in the organism provided by the invention is not influenced by the marked fluorescent dye, has normal biological activity, the marked fluorescent dye is eliminated along with the elimination of the cell, and the change process or the distribution condition of the cell can be accurately reflected by the fluorescence detection result of the cell.

The invention is realized by the following steps:

in a first aspect, embodiments of the present invention provide a cell traceable in vivo, the cell being labelled with a fluorochrome coupled by a chemical bond and bound to the surface of the cell in an irreversible manner.

The cell provided by the embodiment of the invention firstly couples the fluorescent dye with a chemical bond and directly bonds the fluorescent dye on the cell surface in an irreversible manner, so that the cell can be traced or traced in vivo, the marking manner is simple and effective, and the cell is unexpectedly found to be not influenced by the marked fluorescent dye and have normal biological activity, the marked fluorescent dye can be eliminated along with the elimination of the cell, the change process or the distribution condition of the cell can be accurately reflected through the fluorescent detection result of the cell, a new strategy is provided for the detection of the change process or the distribution condition of the exogenous cell in the organism, and a new thought is provided for the research of the pharmacokinetics of the cell treatment medicine in vivo.

In alternative embodiments, the fluorescent dye binds to free amino groups or free thiol groups on the cell surface.

In alternative embodiments, the amino group or the thiol group is an amino acid on the outer surface of the cell membrane of the cell; preferably, the amino acid is selected from arginine, lysine, cysteine.

In alternative embodiments, the amino acid is a protein on the outer surface of the cell membrane of the cell. It should be noted that the protein on the outer surface of the cell membrane may refer to the entire structure of the protein exposed on the outer surface of the cell membrane; it may also refer to a partial structure of a protein partially exposed on the outer surface of a cell membrane, and the rest of the structure is located inside the cell membrane or inside the cell.

In an alternative embodiment, the fluorescent dye is modified with a reactive group through which the fluorescent dye is bound to the amino group or the thiol group on the cell surface;

preferably, the material providing the reactive group is selected from the group consisting of NHS ester, isothiocyanate or maleimide.

In alternative embodiments, the cell is derived from a human or non-human mammal.

In alternative embodiments, the cells are selected from at least one of autologous cells, immune cells, and stem cells;

preferably, the somatic cells are selected from at least one of chondrocytes, hepatocytes, islet cells, and olfactory ensheathing cells;

preferably, the immune cell is selected from at least one of a lymphocyte, a dendritic cell, a monocyte, a macrophage, a granulocyte and a mast cell;

preferably, the stem cell is selected from at least one of an embryonic stem cell, an adult stem cell, a mesenchymal stem cell and an induced pluripotent stem cell.

The cells of the present invention are not limited to cells having a therapeutic action such as somatic cells, immune cells, and stem cells, and may be cells for other non-therapeutic uses, and any cells may be included in the scope of the present invention as long as they are directly modified with a fluorescent dye.

In an alternative embodiment, the fluorescent dye is selected from any one of Alexa Fluor series fluorescent dyes and Cy series fluorescent dyes;

preferably, the Alexa Fluor series fluorescent dye is selected from any one of Alexa Fluor568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633 and Alexa Fluor 647;

preferably, the Cy-series fluorescent dye is any one selected from Cy3, Cy3.5, Cy5, Cy5.5, and Cy 7.

The fluorescent dye of the present invention is not limited to Alexa Fluor-based fluorescent dyes and Cy-based fluorescent dyes, and may be any other type of fluorescent dye, and any fluorescent dye that can be easily observed and labeled because the activated group of the fluorescent dye has no dependency on the selection of the fluorescent dye falls within the scope of the present invention.

In another aspect, an embodiment of the present invention further provides a method for tracking a cell in an organism, including: administering the cell of any of the preceding embodiments to an organism.

In alternative embodiments, the organism is a non-human mammal.

Preferably, the non-human mammal is a mouse, rat, pig, monkey, rabbit, horse, cow, sheep, or dog;

preferably, the tracing method is to detect the fluorescence signal and intensity of the cells.

In an alternative embodiment, the tracing method is for the purpose of diagnosis or treatment of a non-disease.

In another aspect, the present invention also provides a method for preparing a cell according to any one of the previous embodiments, including: contacting the fluorescent dye with the cell to be labeled such that the fluorescent dye is chemically bonded and irreversibly bound to the surface of the cell to be labeled.

In alternative embodiments, contacting the fluorescent dye with the cell to be labeled comprises: reacting the fluorescent dye with the cells to be labeled under weak base conditions.

Preferably, the pH of the weak base conditions is 7.8-8.2.

In alternative embodiments, contacting the fluorescent dye with the cell comprises: mixing a first weak alkaline solution containing the fluorescent dye with a second weak alkaline solution containing the cells to be marked to obtain a mixed solution;

wherein the first weak alkaline solution is PBS or Hanks buffer solution, and the pH value is 7.8-8.2; the second weak alkaline solution is PBS or Hanks buffer solution, and the pH value is 7.8-8.2.

Under the condition of weak base, namely the pH value of the first weak alkaline solution is 7.8-8.2; when the pH value of the second weak alkaline solution is 7.8-8.2, the combination of the fluorescent dye and the cells is facilitated, and the modification efficiency is improved.

In an alternative embodiment, the cells to be labeled in the second weak alkaline solution are cells in a suspension state.

In an alternative embodiment, the concentration of the fluorescent dye in the mixed solution is 10 to 100. mu.M, and the concentration of the cells to be labeled is 1 to 6X 10⁶one/mL.

In an alternative embodiment, the method of making further comprises: and (3) placing the mixed solution under the condition of keeping out of the light for reaction for 10-60 min.

In an alternative embodiment, the method of making further comprises: after the reaction is finished, adding a third alkalescent solution with the volume 2-5 times that of the mixed solution, centrifuging and collecting cells to obtain the cells; wherein the pH of the third weakly alkaline solution is 7.8-8.2.

Preferably, the third alkaline solution is a PBS or Hanks buffer containing glycine at a concentration of 10-200 mM.

The preparation method provided by the embodiment of the invention has simple steps, can efficiently obtain the cells modified by the fluorescent dye, the cells obtained by the preparation method are not influenced by the marked fluorescent dye, have normal biological activity, the marked fluorescent dye can be eliminated along with the elimination of the cells and cannot enter autologous cells of organisms, and the change process or the distribution condition of the modified cells in the bodies can be accurately reflected through the fluorescence detection result.

In a further aspect, embodiments of the present invention also provide a cell medicament comprising as an active ingredient a cell according to any one of the preceding embodiments.

In a further aspect, the use of a cell according to an embodiment of the invention, for example according to any of the preceding embodiments, for the purpose of diagnosis or treatment of a non-disease condition in pharmacokinetic studies.

In an alternative embodiment, the use is for the diagnosis or treatment of a non-disease.

In yet another aspect, the present invention provides a method for modifying a cell for tracking, comprising: the fluorescent dye is coupled with chemical bonds and the surface of the cell is modified in an irreversible combination mode.

The modification method provided by the invention has the advantages that the fluorescent dye is coupled by chemical bonds and is used for modifying the surface of the cell in an irreversible combination mode, and the unexpectedly found that the modified cell can be traced in an organism and has normal biological activity, the marked fluorescent dye can be eliminated along with the elimination of the cell, the random diffusion after the cell is degraded can not influence the accuracy of a fluorescence detection result, and the change process or the distribution condition of the cell can be accurately reflected.

In alternative embodiments, the amino group is from an amino acid of a cell membrane outer surface protein of the cell.

In alternative embodiments, the amino acid is selected from at least one of arginine, lysine, and cysteine.

In an alternative embodiment, the fluorescent dye is selected from any one of Alexa Fluor series fluorescent dyes and Cy series fluorescent dyes.

In an alternative embodiment, the Alexa Fluor series fluorescent dye is selected from any one of Alexa Fluor568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633 and Alexa Fluor 647.

In an alternative embodiment, the Cy-series fluorescent dye is selected from any one of Cy3, Cy3.5, Cy5, Cy5.5, and Cy 7.

In an alternative embodiment, the fluorescent dye is modified with a reactive group through which the fluorescent dye binds to a free amino group or a free thiol group on the cell surface. Wherein, the free amino or free sulfydryl has better reactivity, which is convenient for improving the success rate of chemical coupling.

The free amino groups are derived from arginine and lysine, and the free sulfhydryl groups are derived from cysteine.

In an alternative embodiment, the material providing the reactive group is selected from the group consisting of NHS esters, isothiocyanates, or maleimides.

In an alternative embodiment, the reactive group NHS ester, isothiocyanate is chemically coupled to a free amino group and the reactive group maleimide is chemically coupled to a free thiol group.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1 shows fluorescence intensity of Cy 5-labeled mesenchymal stem cells prepared according to the suspension reaction, the adherence reaction of preparation example 1 of the present invention.

FIG. 2 is a schematic diagram of the cell fluorescent labeling method according to the present invention for cell tracking and pharmacokinetic studies.

FIG. 3 shows the representation of Cy5 labeling efficiency of Cy 5-labeled mesenchymal stem cells prepared according to preparation example 2 of the present invention under reaction conditions of 10. mu.M and 100. mu.M.

Fig. 4 shows the proliferation of cells after cell modification under the reaction condition of 100 μ M of Cy 5-labeled mesenchymal stem cells prepared according to preparation example 2 of the present invention.

Fig. 5 shows the changes of cell phenotype and fluorescence intensity before and after passaging of Cy 5-labeled mesenchymal stem cells prepared according to preparation example 2 of the present invention.

Fig. 6 shows changes in cell phenotype and fluorescence intensity before and after cryopreservation of Cy 5-labeled mesenchymal stem cells prepared according to preparation example 2 of the present invention.

FIG. 7 shows the labeling efficiency of Cy 5-labeled A549 cells, MDCK cells, HepG2 cells, MRC5 cells, UCF cells, RAW 264.7 cells in Experimental example 1 according to the present invention.

FIG. 8 shows the distribution of fluorescent signals in tissues after injection of Cy5-NHS, Cy 5-labeled mesenchymal stem cells according to Experimental example 2 of the present invention.

FIG. 9a shows fluorescence intensity in liver tissue after Cy5-NHS, Cy 5-labeled mesenchymal stem cell injection according to experimental example 2 of the present invention.

FIG. 9b shows fluorescence intensity in lung tissue after Cy5-NHS, Cy 5-labeled mesenchymal stem cell injection according to experimental example 2 of the present invention.

FIG. 10a shows quantitative determination of the number of cells in liver tissue by qPCR method after injection of Cy5-NHS, Cy 5-labeled mesenchymal stem cells according to the experimental example 3 of the present invention.

FIG. 10b shows quantitative determination of cell number in lung tissue by qPCR method after injection of Cy5-NHS, Cy 5-labeled mesenchymal stem cells according to the present invention in Experimental example 3.

FIG. 11 shows the distribution of fluorescent signals in tissues after injection of Cy 5-labeled human-derived pluripotent hepatocytes according to experimental example 4 of the present invention.

FIG. 12a shows fluorescence intensities in liver tissue and spleen tissue after injection of Cy 5-labeled human-derived pluripotent liver cells according to experimental example 4 of the present invention.

FIG. 12b shows quantitative determination of cell number in liver tissue and spleen tissue by qPCR method after injection of Cy 5-labeled human pluripotent liver cells according to experimental example 4 of the present invention.

Fig. 13 shows the distribution of fluorescence signals in tissues after injection of DiR-labeled human mesenchymal stem cells according to experimental example 5 of the present invention.

Fig. 14a shows fluorescence intensities in liver tissue and lung tissue after injection of DiR-labeled human mesenchymal stem cells according to experimental example 5 of the present invention.

Fig. 14b shows the quantitative determination of the cell number in liver tissue and lung tissue by qPCR method after injection of DiR-labeled human mesenchymal stem cells according to experimental example 5 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), biochemistry, which are within the skill of the art. Such techniques are well explained in the literature, e.g. "molecular cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); animal Cell Culture (Animal Cell Culture), ed.R.I. Freshney, 1987, PCR Polymerase Chain Reaction (PCR), ed.polymerase Chain Reaction, Mullis et al, 1994, each of which is expressly incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the devices, formulations, and methodologies that are described in the publications and that might be used in connection with the invention described herein.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the stated limits, ranges excluding either or both of those included limits are also included in the invention.

In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, features and processes that are well known to those of ordinary skill in the art have not been described in order to avoid obscuring the present invention.

The features and properties of the present invention are described in further detail below with reference to examples.

Reagent and medicine

NaOH and glycine were purchased from national pharmaceutical group chemical reagents, Inc.; PBS, physiological saline, Cy5-NHS, DiR Dalian Meiren Biotech Co., Ltd; MTT, dimethyl sulfoxide was purchased from Sigma-Aldrich; FITC labeled CD 29 antibody was purchased from Biolegend; the tissue genome DNA extraction kit is purchased from Guangzhou Meiji Biotechnology Ltd.

The source of animal material, using mouse as model organism, is very close to human regardless of genome composition, individual development, metabolic mode, organ structure, disease pathogenesis, etc. compared with human; therefore, the case where the present invention is applied to a mouse can be applied to other mammals such as a human without any doubt.

Example 1

(1) Process selection for preparing fluorescent dye-labeled cells

(a) Adjusting the pH value of the PBS to 7.8-8.2 by using NaOH solution to obtain weakly alkaline PBS solution for later use; weighing a proper amount of Cy5-NHS powder, and dissolving the Cy5-NHS powder in a weakly alkaline PBS solution; an appropriate amount of glycine powder was weighed and dissolved in weakly alkaline PBS solution at a glycine concentration of 10-200mM, in this example, at a glycine concentration of 100 mM.

(b) And taking the culture dish with the mouse adipose-derived mesenchymal stem cells out of the carbon dioxide incubator, digesting the cells by pancreatin, centrifugally collecting the cells, counting, and resuspending by using weakly alkaline PBS. Adding a proper amount of Cy5-NHS stock solution into the cell suspension, adjusting the concentration of Cy5-NHS to 10 mu M, mixing uniformly, and reacting for 30min at room temperature in a dark place. Obtaining a suspension reaction system with cells in a suspension state.

(c) And taking another culture dish for culturing the mouse fat source mesenchymal stem cells, removing the culture medium, washing the cells once by using PBS, adding 5mL of weakly alkaline PBS into the culture dish, adding a proper amount of Cy5-NHS stock solution, adjusting the concentration of Cy5-NHS to 10 mu M, uniformly mixing, and reacting for 30min at room temperature in a dark place. Obtaining an adherence reaction system with the cells in adherence state.

(e) And after the reaction is finished, respectively adding glycine solution of 5 times of the reaction system into the suspension reaction system and the adherent reaction system, uniformly mixing to terminate the reaction, and reacting the residual unreacted Cy5-NHS with glycine in the glycine solution with a large excess amount. And (3) centrifuging the suspension reaction system to collect cells, resuspending the cells with a proper amount of PBS and counting, and resuspending the cells with the PBS to obtain the mesenchymal stem cells directly marked by the Cy5 suspension reaction. And (3) an adherent reaction system, discarding the supernatant, washing with PBS once, digesting with pancreatin and collecting cells to obtain the mesenchymal stem cells directly marked by the adherent reaction Cy5.

(2) Fluorescence intensity of fluorescently labeled cells

Taking the cultured mesenchymal stem cells from the fat source of the mouse as a blank control, and directly marking the suspension of the mesenchymal stem cells with the Cy5 for suspension reaction and wall-sticking reaction by 1 multiplied by 10⁴One cell/well was inoculated into a black 96-well plate, and the fluorescence intensity of the cells was measured with a microplate reader. The experimental result is shown in fig. 1, the fluorescence intensity of the mesenchymal stem cells directly labeled with Cy5 obtained by suspension reaction is higher than that of the labeled cells obtained by adherence reaction with the same concentration of Cy5-NHS, because the cells are more fully contacted with Cy5-NHS in a suspension state, the reaction sites are more, and the modification amount of Cy5 is more. In addition, the same number of cells, the volume of the adherence reaction system is larger than that of the suspension reaction, and the amount of the reagent is more, so that the suspension reaction system has a better marking effect on the cells, and the cells in the later embodiment are all marked by the suspension reaction system.

Example 2

(1) Optimization method for preparing mesenchymal stem cells marked by fluorescent dye

(a) And taking the culture dish with the mouse adipose-derived mesenchymal stem cells out of the carbon dioxide incubator, digesting the cells by pancreatin, centrifugally collecting the cells, counting, and resuspending by using weakly alkaline PBS.

(b) Dividing the cell suspension into 2 parts in equal volume, respectively adding appropriate amount of Cy5-NHS stock solution, adjusting Cy5-NHS concentration to 10 μ M and 100 μ M respectively, mixing, and reacting at room temperature in dark place for 30 min.

(c) And after the reaction is finished, respectively adding glycine solution which is 5 times of that of the reaction system into the reaction system, uniformly mixing, centrifugally collecting cells, re-suspending the cells by using a proper amount of PBS and counting, and re-suspending the cells by using the PBS to obtain the Cy5 directly-labeled mesenchymal stem cells.

The mesenchymal stem cell obtained in the embodiment is directly labeled with Cy5 fluorescent dye, Cy5 realizes that Cy5 is coupled with the mesenchymal stem cell by chemical bonds and has irreversible combination effect through combination of NHS and amino on cell membrane protein of the mesenchymal stem cell.

By the marking of Cy5, the mesenchymal stem cells can be traced or traced in vivo, and can be applied in vivo, and the change or distribution of the mesenchymal stem cells in vivo can be accurately reflected by the detection of a fluorescent signal (see figure 2).

(2) Characterization of fluorescent labeling efficiency

The proportion of Cy5 positive cells is detected by taking the mesenchymal stem cells derived from mouse fat in culture as a control and using a Cy 5-labeled mesenchymal stem cell up-flow cytometer prepared under the reaction conditions of 10 mu M and 100 mu M. The experimental results are shown in FIG. 3, the Cy5 positive cells reach 97% under the 10. mu.M reaction condition, and the Cy5 positive cells reach 99.9% under the 100. mu.M reaction condition, which indicates that the modification method has higher reaction efficiency. Because the labeling efficiency of the cells is 97% in the 10. mu.M reaction system, part of the cells cannot be labeled, all the cells can be labeled in the 100. mu.M reaction system, the using concentration of the fluorescent dye in the two reaction systems is different by one order of magnitude, the recommended using concentration of the fluorescent dye is not lower than 10. mu.M, and the concentration of Cy5-NHS used in the subsequent experiments is 100. mu.M.

(3) Cy 5-labeled proliferation potency of mesenchymal stem cells

The method comprises the steps of taking cultured mouse adipose-derived mesenchymal stem cells as a blank control, preparing Cy 5-labeled mesenchymal stem cells under the reaction condition of 100 mu M, meanwhile, setting a control group without Cy5-NHS, inoculating the blank control group, the Cy 5-labeled mesenchymal stem cells and the control group cells into a 96-well plate, and examining the influence of modification reaction on cell viability by an MTT method. The experimental result is shown in fig. 4, compared with the blank control group, the 48h survival rate of the Cy 5-labeled mesenchymal stem cells reaches more than 80%, which indicates that the cell modification method does not affect the proliferation capacity of the cells.

(4) Passage stability test

From the fat of the mice in culturePreparing Cy 5-labeled mesenchymal stem cells by using source mesenchymal stem cells as blank control, and labeling the blank control group and Cy 5-labeled mesenchymal stem cells according to the proportion of 1 multiplied by 10⁶Density of individual/well was seeded in 6-well plates with 2 wells seeded with blank control and 1 well seeded with Cy 5-labeled mesenchymal stem cells.

And additionally taking PBS cell suspension containing Cy 5-labeled mesenchymal stem cells into 1 1.5mL centrifuge tubes, putting blank control cells into 2 1.5mL centrifuge tubes, adding FITC-labeled CD 29 antibody into the Cy 5-labeled mesenchymal stem cells and 1 blank control tube respectively, and dyeing for 30min at the temperature of 2-8 ℃ in a dark place. After staining, each group of cells was centrifuged, the cells were resuspended in PBS, and the phenotype of the cells and the proportion of Cy 5-positive cells were examined by flow cytometry.

Cells seeded in 6-well plates were cultured for 48h and collected, stained as described above, and the phenotype of the cells after passaging and the proportion of Cy5 positive cells were examined by flow cytometry.

As shown in fig. 5, the CD 29-positive mesenchymal stem cells account for more than 95% before modification, and after Cy5 modification, the number of Cy 5-positive cells is more than 95%, and the number of CD 29 and Cy5 double-positive cells is also more than 95%, indicating that the cell modification has high efficiency, and the phenotype of the cells is not changed by the cell modification. After cell passage, the Cy5 fluorescence intensity of Cy5 labeled mesenchymal stem cells is more dispersed than that before inoculation, which indicates that the fluorescence intensity of the cells after cell proliferation changes, but the two groups of cells, namely CD 29 and Cy5, are more than 90%, which indicates that the modified cells have passage stability and do not influence the expression of cell surface marker molecules.

(5) Cryopreservation stability detection

Cy 5-labeled mouse adipose-derived mesenchymal stem cells were prepared in a 3X 10 manner⁵Freezing and storing the cell/branch density, and taking the mesenchymal stem cells of the same batch as a blank control group of 6 multiplied by 10⁵And (5) freezing and storing the seeds/stem.

And taking PBS suspension containing blank control and Cy5 labeled mesenchymal stem cells, staining the PBS suspension according to the method CD 29, and detecting the labeling efficiency of Cy5 and the phenotype of the labeled cells by using a flow cytometer to serve as a control before cryopreservation.

Transferring the frozen cells from the ultra-low temperature refrigerator into a liquid nitrogen tank, recovering the cells after the cells are stored in the liquid nitrogen tank for one week, staining blank control and Cy 5-labeled mesenchymal stem cell PBS suspension according to the method CD 29, and detecting the labeling efficiency of Cy5 and the phenotype of the labeled cells by a flow cytometer.

As shown in fig. 6, the experimental results showed that CD 29-positive mesenchymal stem cells reached 98% or more before modification, the number of Cy 5-positive cells was 90% or more after Cy5 modification, and the double-positive cells of CD 29 and Cy5 were 90% or more, which is consistent with the above experimental results. After the cells are frozen and recovered, the fluorescence intensity of Cy5 of the Cy5 marked mesenchymal stem cells is not obviously changed compared with that before the cells are frozen, and the double-positive cells of two groups of cells, namely CD 29 and Cy5, are more than 97 percent, which indicates that the modified cells have freezing stability and do not influence cell phenotype.

Experimental example 1

Cy5 labeling of different types of cells

According to the method, Cy5 labeled human non-small cell lung cancer cells (A549), canine kidney cells (MDCK), human liver cancer cells (HepG2), human embryonic lung cells (MRC5), human umbilical cord-derived primary fibroblasts (UCF) and mouse mononuclear macrophage leukemia cells (RAW 264.7) are prepared under the reaction condition of 100 mu M, and the proportion of Cy5 positive cells is detected by an up-flow cytometer after cell labeling. The experimental results are shown in fig. 7, and after Cy5 modification, the Cy5 positive cells of each group are all above 99%, which indicates that the fluorescence labeling method for cells disclosed in the above embodiment of the present invention is suitable for labeling various cells and has good cell labeling efficiency.

Experimental example 2

In vivo distribution of Cy 5-labeled mesenchymal stem cells

Cy5 labeled human umbilical cord-derived mesenchymal stem cells were prepared, and the concentration of Cy5 in the cell suspension was detected using a microplate reader. According to the concentration of Cy5 in the cell suspension, an appropriate amount of Cy5-NHS solution is taken, mixed with an equal volume of glycine solution, and diluted with PBS to be equal to the concentration of the cell suspension. 12C 57 mice aged 5 weeks were collected at 2X 10⁵Dose/dose Cy 5-labeled mesenchymal stem cellsThe cells were injected into the animals via tail vein, and 4C 57 mice of 5 weeks old were injected into the tail vein with an equal volume of diluted Cy5-NHS solution.

After 30min, 1h, 2h and 8h after injection, 3 mice of Cy 5-labeled mesenchymal stem cell group and 1 mouse of Cy5-NHS group are taken, after euthanasia, heart, liver, spleen, lung and kidney tissues are taken, after physiological saline is washed once, the tissues are laid, and the images are taken under a living body imager of the small animals to examine the distribution condition of fluorescent signals in each tissue. After photographing, the tissue was wrapped with aluminum foil and then stored frozen.

As shown in FIG. 8, the fluorescence signals were mainly distributed in the lung, the fluorescence signals of the Cy5-NHS group were mainly distributed in the liver, and there was no correlation between the two groups in the distribution of the fluorescence signals in the tissues after cell injection. The Cy5 label cannot be dissociated from the cells after the Cy5 labeled mesenchymal stem cells are injected into the body, and the modification has better stability in the body. FIGS. 9a and 9b show the quantification of cells in liver and lung tissue based on fluorescence intensity.

Experimental example 3

Tissue quantification of Cy 5-labeled mesenchymal stem cells

And taking the collected liver and lung tissues of the Cy 5-labeled mesenchymal stem cell group out of a refrigerator, temporarily storing on ice, weighing about 10mg of tissues, and extracting the genomic DNA of the tissues according to an operation manual in the kit. After the extracted genome DNA is quantified, detecting the tissue expression level of the human gene by a qPCR method; meanwhile, setting different numbers of genome DNA control groups extracted from the human umbilical cord source mesenchymal stem cells, and establishing a linear relation between the gene expression level and the number of cells. And quantifying the human mesenchymal stem cells in the liver and kidney tissues at different time points according to the established linear relation.

The experimental results are shown in fig. 10a and 10b, and the results of the quantitative method by the qPCR method are compared with the results of the quantitative method by the fluorescence in fig. 9a and 9b, so that the results of the quantitative method by the tissue and the quantitative results by the fluorescence have the same trend, which shows that the method by the fluorescence directly using the quantitative method has the same reliable accuracy as the method by the qPCR method, and the method by the fluorescence is simpler and more convenient to operate than the qPCR method and is not limited by the species. Due to the convenience and accuracy of the cell modification method, the cell modification method can be used for the pharmacokinetic research of cell therapy medicines.

Experimental example 4

Cy 5-labeled in vivo distribution of proliferatable human-derived pluripotent hepatocytes

Cy 5-labeled proliferatable human-derived pluripotent hepatocytes were prepared by collecting 10 5-week-old C57 mice according to 2X 10⁵One dose/body Cy 5-labeled proliferating pluripotent hepatocytes were injected splenically into animals.

2 mice injected with Cy5 labeled proliferatable bi-potent hepatocytes are randomly selected at 2h, 4h, 8h, 24h and 48h after injection, after euthanasia, heart, liver, spleen, lung and kidney tissues are taken, after physiological saline washing, the tissues are laid out, and the distribution of fluorescent signals in each tissue is examined by taking pictures under a small animal living body imager. After photographing, the tissue was wrapped with aluminum foil and then stored frozen.

The experimental results are shown in fig. 11, after cell injection, the fluorescent signals are mainly distributed in the spleen, and a large number of fluorescent signals are also distributed in the liver tissue, which indicates that a large number of cells enter the liver tissue after intrasplenic injection of the therapeutic cells, and the cells are almost completely eliminated in each organ tissue at 48 h.

FIG. 12a shows the quantitative result of fluorescence intensity in liver and spleen tissues, FIG. 12b shows the analysis of the number of human-derived proliferative liver cells in liver and spleen tissues according to the qPCR method, and both analysis methods show that the distribution of cells in tissues has the same trend, thus demonstrating that the cell fluorescence labeling method provided by the present invention has reliable accuracy in vivo cell tracking as well.

Experimental example 5

In vivo distribution and quantification after endocytosis of dyes

Collecting the cultured human mesenchymal stem cells, adding a proper amount of cell membrane dye DiR into the cell suspension, incubating for 30min in a dark place, washing the cells with PBS, and then resuspending. DiR is a lipophilic fluorescent dye, can enter cells in a free diffusion mode, has remarkably enhanced fluorescence intensity after being combined with cell membranes and intracellular plasma membranes, and is commonly used for marking fine marksCellular, does not affect the activity of the cells, and the marker has no selectivity. 21C 57 mice of 5 weeks old were collected at 1X 10⁵At one dose/one dose, DiR-labeled human mesenchymal stem cells were injected into animals via tail vein.

Randomly taking 3 mice injected with DiR marked human mesenchymal stem cells 15min, 30min, 1h, 2h, 4h, 8h, 24h and 52h after injection, taking heart, liver, spleen, lung and kidney tissues after euthanasia, washing the tissues with physiological saline, placing the tissues, taking pictures under a small animal living body imaging instrument, and observing the distribution condition of fluorescent signals in each tissue. After photographing, the tissue was wrapped with aluminum foil and then stored frozen.

The experimental result is shown in fig. 13, after cell injection, the fluorescence signal is mainly distributed in the lung, and the liver tissue also has a large amount of fluorescence signal distribution, and as time goes on, the fluorescence intensity in the liver tissue is not obviously attenuated, and the fluorescence intensity in the lung tissue is obviously reduced, which is obviously different from the cell distribution result using the cell labeling method of the present invention.

FIG. 14a shows the quantitative result according to the fluorescence intensity in the liver and lung tissues, FIG. 14b shows the quantitative result according to the qPCR method, the quantitative result shows that there is no correlation between the fluorescence intensity in the liver and lung tissues and the cell number in the tissues, and it can be known from the quantitative result (FIG. 14b) of the qPCR method that most of the cells are distributed in the lung tissues after the injection of the cells, the cell numbers of the lung tissue and the liver tissue are greatly different, while FIG. 14a shows that the fluorescence intensities of the liver tissue and the lung tissue are not greatly different, and the fluorescence intensity in the liver tissue begins to rise after 8h, which is supposed to be because the DiR-marked human mesenchymal stem cells are gradually cleared in the animal body after being injected, the DiR is released, the free DiR is gradually distributed in the liver tissue, and possibly into the liver tissue cells, resulting in no significant change in fluorescence intensity in liver tissue up to 52 h.

According to the experimental results, the result of cell tracing by using the endocytosis dye has false positive, and compared with the result, the accuracy of the fluorescent modification tracing method is greatly improved, mainly because the cell endocytosis dye such as DiR is used for marking cells, the marking efficiency is higher, but the marking has no specificity, and the DiR may enter autologous tissue cells after the marked cells enter the body and are metabolized, so that the in-vivo process of the marked cells cannot be accurately reflected. The cell marking method disclosed by the invention couples the fluorescent dye to the protein on the surface of the cell membrane directly through a chemical bond, and the marking is non-reversible, so that the false positive of the fluorescent dye is avoided, and the in-vivo channel distribution of the marked cells can be accurately traced.

In summary, it can be seen that the fluorescence labeling mesenchymal stem cell and the tracing method of the embodiment of the present invention have the following characteristics:

(1) taking mesenchymal stem cells as an example, the fluorescence labeling mesenchymal stem cells prepared by the invention have the cell labeling efficiency of over 95 percent, the cell labeling can not cause toxicity to the cells, the expression conditions of surface marker molecules of the cells before and after cell labeling are not changed, the expression conditions of the surface marker molecules of the cells are not changed after cell inoculation, passage and cryopreservation after labeling, and the fluorescence labeling efficiency is not changed greatly, thereby obtaining unexpected technical effects. The cell fluorescent labeling method is shown to have no influence on cell proliferation, no change in cell phenotype and high passage and freezing stability.

(2) Taking the distribution of human umbilical cord-derived mesenchymal stem cells in a mouse as an example, the mesenchymal stem cells are subjected to fluorescence labeling, a linear relation between fluorescence intensity and cell number is established in vitro, the labeled cells are injected into an animal body, the distribution of fluorescence signals in the animal body after cell injection is detected by a small animal living body imager, the in-vivo process of the cells is traced, and the tissue quantification is carried out on the mesenchymal stem cells based on the fluorescence intensity in the tissue. The cell quantitative method is compared with the qPCR method, which shows that the cell marking method has reliable accuracy, and compared with the qPCR method, the method has the advantages of simple operation, shorter period and more convenient cell tracing.

(3) The detection of in vivo distribution is carried out by adopting other fluorescence labeling modes, the dye is converted into free fluorescent dye and gradually gathered in the liver along with the apoptosis of cells, and the detection result shows false positive by the signal of the free dye in vivo, but the mesenchymal stem cell labeled by the fluorescence can overcome the false positive and accurately show the in vivo process of the cell.

In summary, the surface modification means provided by the invention couples the fluorescent dye molecules to the protein on the surface of the cell membrane through chemical bonds, has better stability, is applied to the temporal distribution of the therapeutic cells after tracer in vivo injection, the marking mode is non-reversible, and when the labeled cells are metabolized in vivo, the fluorescent molecules can be removed along with the cell membrane, so that the in vivo distribution process of the cells can be accurately reflected. In addition, by using the content of the invention, researchers can establish a linear relationship between the fluorescence intensity of cells and the number of cells under in vitro conditions, and can quantitatively detect the number of cells in tissues and blood so as to research the pharmacokinetic properties of the cells. The analysis method has high sensitivity and is not interfered by self signals. The invention provides a method for directly carrying out fluorescence labeling on cells based on chemical bond coupling, carrying out in-vivo cell tracing, and being applied to the in-vivo pharmacokinetics research of cell therapeutic drugs for the first time.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A cell traceable in an organism, wherein said cell is labeled with a fluorescent dye that is coupled via a chemical bond and irreversibly bound to the surface of said cell.

2. The in vivo traceable cell of claim 1, wherein said fluorescent dye binds to a free amino group or a free thiol group on the surface of said cell.

3. The in vivo traceable cell according to claim 2, wherein said amino group or said thiol group is derived from an amino acid on the outer surface of the cell membrane of said cell;

preferably, the amino acid is selected from at least one of arginine, lysine and cysteine.

4. The in vivo traceable cell according to claim 3, wherein said amino acid is derived from a protein on the outer surface of the cell membrane of said cell.

5. The in vivo traceable cell according to any one of claims 2 to 4, wherein said fluorescent dye is modified with a reactive group, and said fluorescent dye is bound to said amino group or said thiol group on the surface of said cell via said reactive group;

6. The cells traceable in vivo according to any one of claims 2 to 4, wherein said cells are in suspension.

7. The in vivo traceable cell according to any one of claims 1 to 4, wherein said cell is derived from a human or non-human mammal; the cells are selected from at least one of autologous cells, immune cells and stem cells;

8. The in vivo traceable cell according to any one of claims 1 to 4, wherein said fluorescent dye is selected from any one of Alexa Fluor series fluorescent dyes and Cy series fluorescent dyes;

9. A method for tracking cells in an organism, comprising: administering the cell of any one of claims 1-8 to an organism.

10. The method of claim 9, wherein the organism is a non-human mammal;

11. A method for producing a cell according to any one of claims 1 to 8, which comprises: contacting the fluorescent dye with the cell to be labeled such that the fluorescent dye is chemically bonded and irreversibly bound to the surface of the cell to be labeled.

12. The production method according to claim 11,

contacting the fluorescent dye with the cell to be labeled comprises: reacting the fluorescent dye with the cells to be labeled under weak base conditions;

preferably, the pH of the weak base conditions is 7.8-8.2;

preferably, contacting the fluorescent dye with the cell comprises: mixing a first weak alkaline solution containing the fluorescent dye with a second weak alkaline solution containing the cells to be marked to obtain a mixed solution; wherein the first weak alkaline solution is PBS or Hanks buffer solution, and the pH value is 7.8-8.2; the second weak alkaline solution is PBS or Hanks buffer solution, and the pH value is 7.8-8.2;

preferably, the cells to be labeled in the second weak alkaline solution are cells in a suspension state.

13. The method according to claim 12, wherein the concentration of the fluorescent dye in the mixed solution is 10 to 100. mu.M, and the concentration of the cells to be labeled is 1 to 6X 10⁶one/mL.

14. The production method according to claim 12 or 13, characterized by further comprising: and (3) placing the mixed solution under the condition of keeping out of the light for reaction for 10-60 min.

15. The method of manufacturing according to claim 14, further comprising: after the reaction is finished, adding a third alkalescent solution with the volume 2-5 times that of the mixed solution, centrifuging and collecting cells to obtain the cells; wherein the pH of the third weak alkaline solution is 7.8-8.2;

16. A cellular medicine, characterized in that it contains, as an active ingredient, the cell according to any one of claims 1 to 8.

17. Use of a cell according to any one of claims 1 to 8 in pharmacokinetic studies.

18. A method for modifying a cell for tracking, comprising: the fluorescent dye is coupled with chemical bonds and carries out surface modification on the suspended cells in an irreversible combination mode.

19. The method of modifying according to claim 18, wherein said fluorescent dye binds to free amino groups or free thiol groups on the surface of said cell;

preferably, said amino group or said thiol group is derived from an amino acid on the outer surface of the cell membrane of said cell;

preferably, the amino acid is selected from at least one of arginine, lysine and cysteine;

preferably, the amino acid is derived from a protein on the outer surface of the cell membrane of the cell.

20. The method of claim 19, wherein the fluorescent dye is modified with a reactive group, and the fluorescent dye is bound to a free amino group or a thiol group on the cell surface via the reactive group;

21. The modification method according to claim 20, wherein the fluorescent dye is selected from any one of Alexa Fluor series fluorescent dyes and Cy series fluorescent dyes;

22. Use of a modification method according to any one of claims 18 to 21 in pharmacokinetic studies.