CN117487739A - Method for purifying 293 cell exosomes on large scale - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for purifying 293 cell exosomes on a large scale, which comprises the following steps: centrifuging the 293 cell culture solution, and discarding the precipitate to obtain a cell culture supernatant; performing first ultrafiltration concentration on the cell culture supernatant by adopting an ultrafiltration membrane bag to obtain first ultrafiltration concentrated solution; performing size exclusion chromatography on the first ultrafiltration concentrate to obtain size exclusion chromatography liquid; and (3) performing second ultrafiltration concentration on the size exclusion chromatography liquid by using hollow fibers to obtain 293 cell exosomes, wherein the filler of the size exclusion chromatography in the step (3) is Smartarose CL-6B. The method provided by the invention can extract 5L 293 cell supernatant in the same batch, obtain the exosomes with high purity, break the bottleneck of large-scale purification of the exosomes, and lay a foundation for subsequent exosome drug loading, engineering and clinical application.

Description

Method for purifying 293 cell exosomes on large scale

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for purifying 293 cell exosomes on a large scale.

Background

The exosomes are nano vesicles secreted by cells, have the diameter of 30-150nm, contain bioactive substances such as proteins, nucleic acids and the like, participate in intercellular communication, and play an important role in the regulation of tumor microenvironment, immune recognition, inflammatory reaction and other physiological and pathological processes. The exosomes can be used as drug delivery vehicles, and are potential and advantageous drug delivery vehicles due to their low toxicity, low immunogenicity, targeting by engineering modifications, etc. However, since the exosomes have small particle size and low abundance of inclusions, whether to obtain high quality exosomes is a key in subsequent studies.

Although methods for isolation and identification of exosomes have evolved, the level of maturation has not been reached. Most of the existing exosome separation technologies are laboratory-level small-scale extraction, including differential centrifugation, tangential flow ultrafiltration, size exclusion, immunoaffinity capture methods and the like, but the methods have the defects of exosomes being destroyed, difficult specific separation, long technical time consumption, difficult large-scale extraction and the like.

293 cells are engineering cells, and have low culture cost, high exosome yield, relatively simple purification process and relatively easy engineering transformation. In the current research, it is more suitable as a drug delivery carrier.

The invention discloses a differential centrifugation-based extracellular body extraction process, which is used for culturing HEK-293 cells, preferably a high-sugar DMEM culture medium, and can effectively solve the problem that the wall-attaching strength of the HEK-293 cells is smaller in the culture process, so that the HEK-293 cells in the culture medium are produced at the same position, and the HEK-293 cells are conveniently separated.

Chinese patent application 202010094234.2 discloses a method for preparing a drug-loaded exosome comprising the steps of: step (1): culturing and reforming HEK293T cells by using a culture medium without exosomes and collecting the cells; step (2): HEK293T cells are placed in a substrate with a plurality of channels with the diameters of 200-600nm for culture, then the substrate is placed in a buffer solution of target plasmid DNA, electroporation is carried out first, and then directional current stimulation is carried out for 8-12 hours; step (3): and (3) carrying out ultracentrifugation on the obtained buffer solution, collecting precipitate and carrying out resuspension by using the buffer solution to obtain the exosomes loaded with the gene medicine.

There is a need in the art for methods of purifying 293 cell exosomes on a large scale.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides the following technical scheme:

it is an object of the present invention to provide a method for large scale extraction of 293 cell exosomes, the method comprising the steps of:

(1) Centrifuging the 293 cell culture solution, and discarding the precipitate to obtain a cell culture supernatant;

(2) Performing first ultrafiltration concentration on the cell culture supernatant by adopting an ultrafiltration membrane bag to obtain first ultrafiltration concentrated solution;

(3) Performing size exclusion chromatography on the first ultrafiltration concentrate to obtain size exclusion chromatography liquid;

(4) Performing second ultrafiltration concentration on the size exclusion chromatography liquid by adopting hollow fibers to obtain 293 cell exosomes;

the size exclusion chromatography packing material in step (3) was Smartarose CL-6B.

In some embodiments, the ultrafiltration membrane packets in step (1) include, but are not limited to, hydrophilic Polyethersulfone (PES) membrane packets, polypropylene (PP) membrane packets, polyvinylidene fluoride (PVDF) membrane packets, polyacrylonitrile (PAN) membrane packets, polyvinylchloride (PVC) membrane packets, or Polyethersulfone (PES) membrane packets.

In some preferred embodiments, the ultrafiltration membrane packet in step (1) is a Polyethersulfone (PES) membrane packet.

In some embodiments, the ultrafiltration membrane packet in step (1) has a cutoff of at least 100KD.

In some embodiments, the ultrafiltration membrane packet in step (1) may have a cutoff of 100KD to 300KD.

In some embodiments, the amount of the ultrafiltration membrane packets in step (1) can be 100KD, 110KD, 120KD, 130KD, 140KD, 150KD, 160KD, 170KD, 180KD, 190KD, 200KD.

In some preferred embodiments, the ultrafiltration membrane packet in step (1) may have a cutoff of 100KD.

In some embodiments, the hollow fibers in step (4) comprise hydrophilic Polyethersulfone (PES) hollow fibers, polypropylene (PP) hollow fibers, polyvinylidene fluoride (PVDF) hollow fibers, polyacrylonitrile (PAN) hollow fibers, polyvinylchloride (PVC) hollow fibers, or Polyethersulfone (PES) hollow fibers.

In some preferred embodiments, the hollow fibers in step (4) are Polyethersulfone (PES) hollow fibers.

In some embodiments, the hollow fibers of step (4) have a cut-off of at least 100KD.

In some embodiments, the hollow fibers in step (4) may have a retention of 100KD to 300KD.

In some embodiments, the amount of retention of the hollow fibers in step (4) may be 100KD, 110KD, 120KD, 130KD, 140KD, 150KD, 160KD, 170KD, 180KD, 190KD, 200KD.

In some preferred embodiments, the retention of the hollow fibers in step (4) may be 100KD.

In some embodiments, the ultrafiltration concentrate in step (2) has a volume of 5 to 15 volumes.

The volume is relative to the original liquid volume, i.e., the volume of the cell culture supernatant of step (1).

In some preferred embodiments, the ultrafiltration concentrate in step (2) has a volume of 5 to 10 volumes.

The volume is relative to the original liquid volume, i.e., the volume of the cell culture supernatant of step (1).

In some embodiments, step (2) further comprises ultrafiltration concentrating the post-change fluid.

In some embodiments, the volume of the post-ultrafiltration concentrate is 3-5 volumes.

In some preferred embodiments, the volume of the post-ultrafiltration concentrate is 5 volumes.

The volume is relative to the original liquid volume, i.e., the volume of the cell culture supernatant.

The second object of the present invention is to provide an exosome obtained by any of the above methods.

It is a further object of the present invention to provide a use of said exosomes for delivering a drug and/or for preparing a drug.

The inventor provides a large-scale separation and purification method for exosomes through a large number of screening, wherein the selected size exclusion chromatography packing Smartarose CL-6B can effectively separate the hybrid protein, the content of the purified exosomes is obviously improved, the yield of the obtained exosomes is up to 9E+10 particle numbers, and the purity is up to 5E+8 parts ics/mug. The method provided by the invention not only can extract 5L 293 cell supernatant in the same batch, but also can obtain higher exosome purity, breaks through the bottleneck of large-scale exosome purification, and lays a foundation for subsequent exosome drug loading, engineering and clinical application.

Drawings

FIGS. 1-3 show the electron microscopy results of the purified 293 cell exosomes of the present invention.

FIG. 4 shows the particle size, particle concentration, BCA protein concentration and purity of the purified 293 cell exosomes of the present invention.

FIGS. 5-6 show the results of detection of the purified 293 exosome surface markers of the present invention.

FIG. 7 shows the detection results of the markers within the 293 exosome vesicles purified according to the invention.

FIGS. 8-12 show exosome content and protein concentration, respectively, in 293 cell exosomes purified using Smartarose CL-4B, smartarose 4FF, smartarose 6FF, smac Core700 and Diamond Layer 700 fillers, wherein the bar graphs represent exosome content and the curves represent hetero-protein content.

FIG. 13 shows exosome content and protein concentration in 293 cell exosomes purified using Smartarose CL-6B packing, wherein the bar graph represents exosome content and the curve represents hetero-protein content.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

Numerical ranges as used herein should be understood to have enumerated all numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

As used herein, the term "include" or "comprising" means "including but not limited to. The term is intended to be open ended to specify the presence of any stated features, elements, integers, steps, or components, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more limiting terms "consisting of … …" and "consisting essentially of … …". In one embodiment, the term "comprising" as used throughout the application, and in particular in the claims, may be replaced by the term "consisting of … …".

As used herein, the terms "optional," "any," or "any" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1 antibody heavy chain variable region" means that an antibody heavy chain variable region of a particular sequence may be, but is not required to be, present. As used herein, "a" and "an" are used in this disclosure to refer to one or more than one grammar object.

As used herein, the term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

The buffer used in the present invention:

equilibration buffer 1: phosphate buffer, prepared with commercial PBS powder, pH7.4, conductance 16mS/cm;

equilibration buffer 2:120mmol/L disodium hydrogen phosphate+sodium dihydrogen phosphate, pH7.4, conductance 14mS/cm;

cleaning buffer 1:0.5mol/L sodium hydroxide, pH 12, conductance 95mS/cm;

cleaning buffer 2:1mol/L sodium hydroxide, pH 12, conductance 80mS/cm;

preservation solution: 20% ethanol.

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention. Such structures and techniques are also described in a number of publications, such as the molecular cloning laboratory guidelines (fourth edition) (Cold spring harbor laboratory Press), ausubel, F.M et al, current Protocols in Molecular Biology, greene Publishing Assoc, and Wiley-lnterscience.

EXAMPLE 1 preparation of 293 cell exosomes

(1) Cell culture supernatants were harvested by centrifugation: 293 cells (volume) are harvested after 5 to 8 days of culture in a bioreactor, centrifuged for 10 minutes under 300 Xg by a floor type centrifuge, then centrifuged for 10 minutes under 2000 Xg, and bottom sediment is discarded after centrifugation is finished to obtain transparent and clear 5L cell culture supernatant containing exosomes;

(2) Ultrafiltration, concentration and liquid exchange of 100 KD: the cell culture supernatant was subjected to tangential flow ultrafiltration using an ultrafiltration membrane pack (Kebaite, CAT#: HFELA 01001060P) of hydrophilic Polyethersulfone (PES) material having a retention of 100kD. Using 55L/m ² The membrane package and the pipeline are flushed by the purified water, and the transmembrane pressure (Transmembrane Pressure, TMP) is maintained to be 0-1bar, and the conductance of the effluent from the permeation end is lower than 50 mu m/cm; rinsing the membrane package and the pipeline by using a balance buffer solution 1, maintaining TMP at 0-1bar until the pH and the conductivity of the permeate end effluent are consistent with those of the balance buffer solution 1; adding the above cell culture supernatant, maintaining TMP at 0-1bar, concentrating 5-10 times volume (relative to original liquid volume, i.e. 5L cell culture supernatant); using an equilibrium buffer 1, carrying out equal volume liquid exchange according to the volume of the concentrated sample, maintaining TMP at 0-1bar, and changing the liquid by 5 times of volume (relative to the original liquid volume, namely 5L of cell culture supernatant); after the liquid change, the samples were taken out entirely, and the TMP was maintained at 0-1bar using cleaning buffer 1, and the membrane bag and the tubing were cleaned.

(3) Size exclusion chromatography: the cell culture supernatants after the above concentrated changes were purified using size exclusion chromatography medium Smartarose CL-6B (Tiandi Kong, cat#: SEC 0093). Connecting the chromatographic Column filled with the chromatographic medium to a protein purifier, flushing the chromatographic Column with purified water by 3 Column Volumes (CV); equilibration of the column 3CV using equilibration buffer 2; equilibration of the column 3CV using equilibration buffer 1; sampling the concentrated cell culture supernatant after liquid exchange, setting the sampling flow rate at 1mL/min, and starting to collect exosome-containing components after A280 is more than 100mAU until A280 is reduced below 100mAU, and stopping collecting; after the sample injection is finished, balancing the chromatographic column 2CV by using a balancing buffer solution 1, and stopping collecting the flow through after 1.5CV; back-flushing the column with clean buffer 2 at 1.5CV; equilibration of the column 3CV using equilibration buffer 2; equilibration of the column 3CV using equilibration buffer 1; washing 1.5CV of the chromatographic column by using a preservation solution to preserve the chromatographic column; except that the cleaning buffer 2 is used for back flushing the chromatographic column, the buffer and the sample in the rest steps flow through the chromatographic column in the forward direction.

(4) Ultrafiltration concentration of 100KD hollow fiber: the flow-through component containing the exosome is subjected to ultrafiltration concentration by adopting a hollow fiber ultrafiltration membrane bag (Kebaite, CAT#: HFELA 01001060P) made of hydrophilic Polyethersulfone (PES) with a retention amount of 100KD, and the concentration is 5-10 times of the volume, so that a high-purity and high-concentration exosome sample is obtained.

Example 2 identification of 293 exosomes

The exosomes obtained in example 1 were identified.

The result shows that the particle size peak of the 293 exosome is 83.2nm and the particle concentration is 1.40E+11particle/mL (particle size and particle concentration are determined by a nanofluidic instrument); BCA protein concentration was 0.69. Mu.g/. Mu.L; exosome purity was 2.03E+08 particle/. Mu.g protein (FIGS. 1-4).

The Western-Blotting method is used for detecting the surface marker, and the identification result is as follows: CD9 positive, CD81 positive (fig. 5-6); the detection of the intra-vesicle markers using the Western-Blotting method resulted in the following identification: alix positive (fig. 7).

Comparative example 1

The size exclusion chromatography packing of example 1 was replaced with Smartarose CL-4B (Tiandi Kong, cat#: SEC 0083) and the remaining parameters and steps were the same as in example 1.

Comparative example 2

The size exclusion chromatography packing of example 1 was replaced with Smartarose 6FF (sum of heaven and earth, CAT#: SEC 0113) and the remaining parameters and steps were the same as in example 1.

Comparative example 3

The size exclusion chromatography packing of example 1 was replaced with Smartarose 4FF (Tiandi, cat#: SEC 0103) and the remaining parameters and steps were the same as in example 1.

Comparative example 4

The size exclusion chromatography packing of example 1 was replaced with Smac Core700 (Tiandi and CAT#: SEC 0282) and the remaining parameters and steps were the same as in example 1.

Comparative example 5

The size exclusion chromatography packing of example 1 was replaced with Diamond Layer 700 (Bogurone, CAT#: AI 0461) and the remaining parameters and steps were the same as in example 1.

The exosomes purified in example 1 and comparative examples 1 to 5 were tested.

The above six kinds of packing were screened using 10mL columns of the same shape and the same column volume. The column packing was performed according to the method recommended in the packing specification. Before use, 1mL of the cell supernatant concentrated by the above procedure was equilibrated with PBS, then eluted with PBS, and the effluent was collected in one fraction per 500 μl and tested for exosomes and proteins content of the fraction.

Exosome content/concentration was obtained using nanofluidic assay and protein content was performed by BCA kit.

Exosome purity can be calculated by exosome content and protein concentration:

purity of exosomes (particales/μg) =exosome concentration (particales/μl)/protein content (μg/μl)

The separation column was thus evaluated for the yield and purity of exosome purification.

The results showed that Smartarose 6FF,Smartarose 4FF and Smartarose CL-4B fillers failed to completely separate exosomes from the hybrid protein, resulting in lower yields of exosomes collected to 1E+8particles/μg, 5.4E+9 particle count, 2E+10 particle count and 4.8E+9 particle count, respectively (FIGS. 8-10); smac Core700 and Diamond Layer 700 fillers are based on a composite mode, can effectively adsorb hybrid proteins, but only harvest the purified exosomes with a lower content of 6.5E+8 particles and 5.2E+9 particles (FIGS. 11 and 12); the Smartarose CL-6B packing adopted by the invention has separation capability, and the exosome yield is up to 9E+10 particle number (figure 13) and the purity is up to 5E+8 particles/mug.

Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims (15)

1. A method for large scale extraction of 293 cell exosomes, comprising the steps of:

(1) Centrifuging the 293 cell culture solution, and discarding the precipitate to obtain a cell culture supernatant;

(2) Performing first ultrafiltration concentration on the cell culture supernatant by adopting an ultrafiltration membrane bag to obtain first ultrafiltration concentrated solution;

(3) Performing size exclusion chromatography on the first ultrafiltration concentrate to obtain size exclusion chromatography liquid;

(4) Performing second ultrafiltration concentration on the size exclusion chromatography liquid by adopting hollow fibers to obtain 293 cell exosomes;

the size exclusion chromatography packing material in step (3) was Smartarose CL-6B.

2. The method of claim 1, wherein the ultrafiltration membrane packet in step (1) comprises a hydrophilic Polyethersulfone (PES) membrane packet, a polypropylene (PP) membrane packet, a polyvinylidene fluoride (PVDF) membrane packet, a Polyacrylonitrile (PAN) membrane packet, a polyvinyl chloride (PVC) membrane packet, or a Polyethersulfone (PES) membrane packet.

3. The method of claim 2, wherein the ultrafiltration membrane packet in step (1) is a Polyethersulfone (PES) membrane packet.

4. A method according to any one of claims 1 to 3, wherein the ultrafiltration membrane packets in step (1) have a cutoff of 100KD to 300KD.

5. The method of claim 4, wherein the ultrafiltration membrane packet in step (1) has a cutoff of 100KD.

6. The method of claim 5, wherein the hollow fibers in step (4) comprise hydrophilic polyethersulfone hollow fibers, polypropylene hollow fibers, polyvinylidene fluoride hollow fibers, polyacrylonitrile hollow fibers, polyvinyl chloride hollow fibers, or polyethersulfone hollow fibers.

7. The method of claim 6, wherein the hollow fibers in step (4) are polyethersulfone hollow fibers.

8. The method of claim 7, wherein the hollow fibers in step (4) comprise hydrophilic polyethersulfone hollow fibers, polypropylene hollow fibers, polyvinylidene fluoride hollow fibers, polyacrylonitrile hollow fibers, polyvinyl chloride hollow fibers, or polyethersulfone hollow fibers.

9. The method of claim 8, wherein the hollow fibers in step (4) are polyethersulfone hollow fibers.

10. The method according to claim 9, wherein the hollow fibers in step (4) have a retention of 100KD to 300KD.

11. The method of claim 10, wherein the hollow fibers in step (4) have a retention of 100KD.

12. The method of claim 11, wherein the ultrafiltration concentrate in step (2) has a volume of 5 to 15 volumes;

the volume is relative to the original liquid volume, i.e., the volume of the cell culture supernatant of step (1).

13. The method of claim 12, wherein the ultrafiltration concentrate in step (2) has a volume of 5 to 10 volumes;

the volume is relative to the original liquid volume, i.e., the volume of the cell culture supernatant of step (1).

14. Exosomes obtainable by the method according to any one of claims 1 to 13.

15. Use of an exosome according to claim 14 for delivering a drug and/or for preparing a drug.