CN115747049A - Separation and purification device for stem cell exosomes and paracrine active macromolecules and application method thereof - Google Patents
Separation and purification device for stem cell exosomes and paracrine active macromolecules and application method thereof Download PDFInfo
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Abstract
The invention discloses a separation and purification device for stem cell exosomes and paracrine active macromolecules and a use method thereof. The invention organically combines tangential flow microfiltration, tangential flow ultrafiltration and magnetic bead adsorption, integrates a set of separation system, separates and purifies exosomes and active macromolecules in cell culture solution, overcomes the defects of a single method, further strengthens the advantages of the single method, and improves the purity and extraction efficiency of products.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a separation and purification device for stem cell exosomes and paracrine active macromolecules and a using method thereof.
Background
Stem cell therapy has proven to be of great clinical value as a popular research direction in recent years. Among them, mesenchymal stem cells are widely used in clinical studies of various diseases due to their wide source, strong regenerative capacity, and excellent immunoregulatory ability. At present, the mesenchymal stem cells are generally considered to play a main role through paracrine, and the stem cell paracrine factor therapy is taken as a cell-free treatment mode, greatly reduces the risks of rejection, ectopic differentiation and the like after xenotransplantation, and has remarkable effects in the research of a plurality of fields such as wound repair, ischemic diseases, COVID-19 treatment, medical science and beauty, facial reshaping and the like. The paracrine factors of stem cells for therapeutic or medical and cosmetic purposes mainly comprise exosomes and biological macromolecules such as various growth factors and trophic factors. Although the paracrine factor of stem cells has shown great potential in the research field, the components are complex, the yield is low, and the separation and purification efficiency is low, so that the application of the paracrine factor in clinic is greatly limited.
Exosomes carry important biomolecules from maternal cells, including RNA, proteins, lipids, metabolites and other molecules, which play a crucial role in cell signaling, cell physiology and pathology, and the like. In recent years, the method has become a hot research point in the field of stem cell therapy, and has a remarkable treatment effect in the treatment of various diseases. However, many biological structures with similar structures but different functions, such as microvesicles (100 to 1000 nm) and apoptotic bodies (50 to 4000 nm), are generated along with exosomes in the physiological activities of cells. Therefore, the identification and control of key quality attributes (e.g., size, purity, molecular composition, markers, etc.) of exosome products will be key to influencing their potency and stability, as well as an important factor in determining whether they are ultimately successfully applied in clinical practice. Currently, the extraction of exosomes mainly comprises a differential ultracentrifugation method, a density gradient centrifugation method, an ultrafiltration method, a polymer precipitation method and an immunoaffinity separation method, and the separation principles are different from each other, and are different from each other in the aspects of product extraction efficiency, purity, recovery rate and extraction cost. The main methods for separating and purifying the macromolecular active factors secreted by the stem cells are ultrafiltration, dialysis and the like, and the aims of desalting, decoloring and the like of the product are mainly fulfilled.
Among the above methods, ultrafiltration is the most suitable method for scale-up production, and currently, tangential flow ultrafiltration is the most widely used method, which can treat a large amount of culture solution and has high separation efficiency. However, due to the limitation of the ultrafiltration method based on the principle of particle size separation, the method has a particle "squeezing effect", which causes the concentration speed of the exosome to be slower and slower, and finally a product with high concentration cannot be obtained, and generally needs to be further concentrated by an ultracentrifugation method, and the total recovery rate is low. The immunoaffinity separation method, a typical method is an immunomagnetic bead separation method, and is the most suitable method for extracting exosomes in small batches in a laboratory at present. Based on the exosome surface specificity marker protein or membrane structure molecules, the exosome in the culture solution is directly adsorbed by using corresponding antibody cross-linked magnetic beads, and then is eluted by using eluent, so that the exosome with high purity is obtained, and the extraction speed is high. And exosome subtypes can be isolated by selecting different antibodies. However, the method has low treatment capacity, and the magnetic beads and the culture solution are required to be mixed in a high proportion so as to be well adsorbed. The magnetic beads are expensive and not suitable for processing a large amount of culture solution.
However, no more perfect method exists for separating and purifying the macromolecular active factors secreted by stem cells. Mainly, the complete cell culture medium, especially the culture medium added with serum or serum substitutes, also contains more biological macromolecules, which are difficult to be completely distinguished from the biological macromolecules secreted by the cells.
Disclosure of Invention
The invention discloses a separation and purification device for stem cell exosomes and paracrine active macromolecules and a using method thereof, aiming at the problems that in the prior art, no separation and purification device simultaneously applied to separation of the stem cell exosomes and the paracrine active macromolecules and low separation and extraction speed exist, and higher recovery rate and higher extraction speed are obtained.
The invention is realized by the following technical scheme:
a separation and purification device for stem cell exosomes and paracrine active macromolecules comprises a main control panel device, a liquid storage device and a liquid storage hanging rack;
the main control panel device comprises a touch operation screen, a pretreatment device, an ultrafiltration purification device and a suction device, wherein the liquid storage device comprises a raw liquid cylinder base and a filtrate cylinder base and is used for fixing a raw liquid cylinder and a filtrate cylinder;
the pretreatment device comprises a filter fixing frame and a pretreatment pump, wherein the filter fixing frame is used for fixing a hollow fiber microfiltration membrane, a stock solution cylinder is connected with the pretreatment pump and a hollow fiber microfiltration membrane pipeline, the hollow fiber microfiltration membrane is connected with the stock solution cylinder pipeline, and the hollow fiber microfiltration membrane is connected with a filtrate cylinder pipeline and is used for collecting filtrate;
the ultrafiltration purification device comprises a circulating pump, a hollow fiber ultrafiltration membrane fixing frame, a magnetic plate, a magnetic bead container fixing clamp and a power pressure regulator, wherein the hollow fiber ultrafiltration membrane fixing frame is used for fixing a hollow fiber ultrafiltration membrane, the magnetic plate and the magnetic bead container fixing clamp are used for fixing a low-adsorption magnetic bead container, a filtrate cylinder is connected with the circulating pump and the hollow fiber ultrafiltration membrane through pipelines, the hollow fiber ultrafiltration membrane is connected with the power pressure regulator through a pipeline, a selective pipeline between the hollow fiber ultrafiltration membrane and the power pressure regulator is connected with the low-adsorption magnetic bead container, and the power pressure regulator is connected with the filtrate cylinder through a pipeline;
the suction device comprises a suction barrel, a suction barrel electric propeller, a suction barrel, a fixed groove and an electric propulsion sliding rail, wherein the lower edge of the suction barrel is fixed, the lower edge of the suction barrel is fixed with the electric propulsion sliding rail, the suction barrel is connected with a filtrate cylinder and an ultrafiltration purification device through pipelines.
Further, the ultrafiltration purification devices are 1 to 3 groups and are connected in series or in parallel.
Further, the hollow fiber microfiltration membrane comprises a liquid inlet and a liquid outlet, wherein a filtrate port I is connected with a filtrate cylinder; the hollow fiber ultrafiltration membrane comprises a liquid inlet II, a liquid outlet II, a filtrate port III and a filtrate port IV; the low-adsorption magnetic bead container comprises a container cover, a container bin, a liquid inlet III and a liquid outlet III, wherein a liquid separation baffle is arranged in the container bin, and a liquid outlet screen is arranged at the liquid outlet III; the suction barrel comprises a suction pipe, a piston push-pull rod and a liquid suction port.
Furthermore, a main pipeline electric liquid flow detection clamp is arranged on a main pipeline of the electric pressure regulator in the ultrafiltration purification device, which leads to the suction barrel and the filtrate cylinder, a loop electric liquid flow detection clamp is arranged on a loop pipeline between the ultrafiltration purification device and the filtrate cylinder, and a suction pipeline liquid flow detection clamp is arranged on a pipeline between the ultrafiltration purification device and the suction barrel.
Furthermore, liquid level detectors are arranged in the stock solution cylinder and the filtrate cylinder.
Furthermore, the pipelines used for connecting the devices are silica gel pipelines.
The use method of the device for separating and purifying the stem cell exosomes and the paracrine active macromolecules comprises the following steps:
the method for separating and purifying the stem cell exosomes comprises the following steps:
1) Installing and fixing a separation and purification device for the stem cell exosomes and paracrine active macromolecules, installing and fixing a stock solution cylinder, a filtrate cylinder, a hollow fiber microfiltration membrane, a hollow fiber ultrafiltration membrane, a low adsorption magnetic bead container and a suction barrel, communicating the stock solution cylinder, the filtrate cylinder, the hollow fiber microfiltration membrane, the hollow fiber ultrafiltration membrane, the low adsorption magnetic bead container and the suction barrel with pipelines, placing a culture solution rich in the stem cell exosomes into the stock solution cylinder, respectively fixing a buffer solution and an eluent on a hook, and respectively adopting a hanging rack pipeline electric clamp to control the buffer solution and the eluent to flow into the filtrate cylinder;
2) Starting a pretreatment pump, filtering culture solution rich in stem cell exosomes in a stock solution cylinder by a hollow fiber microfiltration membrane, and collecting filtrate to a filtrate cylinder;
3) Step 2) after the pretreatment is finished, opening a circulating pump and adjusting the pressure of a pressure regulator, so that the liquid in a filtrate cylinder is sequentially subjected to ultrafiltration and affinity adsorption through a hollow fiber ultrafiltration membrane and a low adsorption magnetic bead container, the treated filtrate enters the filtrate cylinder and is subjected to ultrafiltration and affinity adsorption in a circulating manner, and a buffer solution is supplemented into the filtrate cylinder in the ultrafiltration and affinity adsorption process through an electric clamp of a rack pipeline corresponding to the control buffer solution;
4) Step 3) after the ultrafiltration and affinity adsorption treatment is finished, closing the circulating pump, opening a hanger pipeline electric clamp corresponding to the eluent, adding the eluent into the filtrate cylinder, closing the hanger pipeline electric clamp, starting the circulating pump for elution, eluting exosomes adsorbed on the magnetic beads, and closing the circulating pump after the elution is finished;
5) After the elution is finished, the electric propeller is started, and liquid in the filtrate cylinder and the pipeline system is sucked into the suction barrel to complete the separation and purification of the exosome;
the method for separating and purifying the paracrine active macromolecule comprises the following steps:
1) Installing and fixing a stem cell exosome and paracrine active macromolecule separation and purification device, installing and fixing a stock solution cylinder, a filtrate cylinder, a hollow fiber microfiltration membrane, a hollow fiber ultrafiltration membrane and a suction barrel, placing a culture solution rich in paracrine active macromolecules into the stock solution cylinder, fixing a buffer solution on a hook, and controlling the buffer solution to flow into the filtrate cylinder by adopting a hanging rack pipeline electric clamp;
2) Starting a pretreatment pump, filtering the culture solution rich in paracrine active macromolecules in the stock solution tank through a hollow fiber microfiltration membrane, and collecting filtrate to a filtrate tank;
3) Step 2) after the pretreatment is finished, opening a circulating pump and adjusting the pressure of a pressure regulator to ensure that the liquid in a filtrate cylinder is ultrafiltered by a hollow fiber ultrafiltration membrane, feeding the treated filtrate into the filtrate cylinder, circularly performing ultrafiltration, and supplementing a buffer solution into the filtrate cylinder in an ultrafiltration process by controlling an electric clamp of a hanging rack pipeline corresponding to the buffer solution;
4) After ultrafiltration is finished, the electric propeller is started, liquid in the filtrate tank and the pipeline system is sucked into the suction barrel, and separation and purification of paracrine active macromolecules are completed.
Furthermore, the magnetic beads in the low-adsorption magnetic bead container are streptomycin affinity magnetic beads and are cross-linked with a biotin-labeled Tim4 antibody.
In the invention, the culture solution rich in paracrine active macromolecules and the culture solution rich in paracrine active macromolecules are obtained by a continuous production method, and cells are cultured by adopting a hollow fiber membrane bioreactor 3D, so that on one hand, the culture space of the cells is closer to a three-dimensional microenvironment in an organism, and secreted exosomes and active macromolecules are closer to the physiological activity of the secreted exosomes and the active macromolecules in the organism; on the other hand, the cell culture space and the culture medium are divided by the ultrafiltration membrane, so that exosomes secreted by cells and exosomes carried in the culture medium are effectively isolated, and the culture medium without using the exosomes is not limited; the biological macromolecules in the culture medium or the biological macromolecules secreted by the cells can permeate the ultrafiltration membrane; according to the continuous production method, different products are cultured and enriched in stages, culture solution is harvested in stages, exosome production is carried out in a cell growth vigorous stage, the normal physiological function of exosome is effectively guaranteed, 3D culture is carried out, cells grow in a stacked mode, the cell amplification stage is far higher than that of 2D culture, paracrine bioactive macromolecule production is carried out when the number of the cells reaches saturation, and the yield of active macromolecules is effectively increased.
The invention relates to a continuous production method of stem cell exosomes and paracrine active macromolecules, which specifically comprises the following steps:
(1) 3D culture of the hollow fiber membrane bioreactor: resuspending the stem cells subjected to in vitro subculture by using a complete culture medium, inoculating the stem cells to the external space of the hollow fiber membrane bioreactor, connecting a complete culture medium storage tank, starting a circulating pump, and replacing the culture medium when the glucose consumption is more than 80%;
(2) When the consumption speed of the culture medium tends to be stable, harvesting a culture solution rich in the exosomes of the stem cells from the external space of the hollow fibers, filling the external space of the hollow fibers with a new complete culture medium, and repeating the step until the consumption speed of the culture medium starts to be obviously reduced and the cell density of the external space of the hollow fibers is saturated;
(3) Replacing a basic culture medium without biological macromolecules for culture, collecting culture solution rich in the paracrine active macromolecules of the stem cells from a storage tank at intervals, replacing the new basic culture medium in an equivalent manner, and repeating the step for 2 to 3 times;
(4) And (4) extracting, separating and purifying the stem cell exosome culture solution and the stem cell paracrine active macromolecule culture solution obtained in the step (2) and the step (3) respectively to obtain the stem cell exosome and the stem cell paracrine active macromolecule.
After the stem cell exosome culture solution obtained in the step (2) is subjected to low-temperature centrifugal separation treatment, cell debris and protein precipitation are removed to obtain supernatant for later use; and (4) centrifuging the stem cell paracrine active macromolecule culture solution obtained in the step (3) to remove cell debris to obtain supernatant for later use. The complete medium is a mesenchymal stem cell serum-free basal medium +5% serum substitute, and the complete medium is cultured under the conditions of 37 deg.C and 5% CO 2 (ii) a The basal medium had a composition of DMEM/F12+1.5% D-glucose + 50. Mu. Mol/L L-ascorbic acid, and the culture conditions were 37 ℃ and 5% CO in the case of the basal medium 2 ，5% O 2 (ii) a In the step (2), the volume of the complete medium to be replaced each time is gradually increased according to the glucose consumption rate.
According to the invention, the exosome is extracted by synchronously carrying out hollow fiber ultrafiltration (ultrafiltration) and low adsorption magnetic beads (affinity adsorption), the volume of the separated liquid can be rapidly reduced in the early stage of separation, the concentration of the exosome is continuously increased, the affinity adsorption magnetic beads are more and more efficient in adsorbing the exosome, the exosome is adsorbed on the surface of the magnetic beads, the content of free exosome in the separated liquid is effectively reduced, the extrusion effect in the ultrafiltration process is greatly reduced, and the blockage of the ultrafiltration membrane and the loss of the exosome are effectively avoided; the method overcomes the defects of slower and slower separation speed and product loss of an ultrafiltration method, overcomes the defects of incapability of processing large-volume samples and high cost of an affinity adsorption magnetic bead method, and obviously improves the extraction speed and the recovery rate.
The invention can obviously reduce the cost of consumables, the hollow fiber ultrafiltration membrane used by the device can be repeatedly used for more than ten times through a simple automatic washing process without replacement or back washing, and the affinity magnetic beads can be repeatedly used for more than five times without repeated filling.
According to the invention, the separation and purification device for the stem cell exosomes and paracrine active macromolecules is an automatic device, and integrates the functions of hollow fiber membrane microfiltration, hollow fiber membrane ultrafiltration, magnetic bead affinity adsorption, product cleaning, product elution, product automatic suction and the like, so that the automatic extraction and modular design of the exosomes and the stem cell paracrine active macromolecules can be realized, the separation stages or combination modes can be increased independently, the manual operation steps are greatly reduced, and the product quality stability is improved.
When the stem cell exosome is extracted, the exosome or the cross-linked antibody adsorbed on the surface of the magnetic bead is effectively prevented from being washed away by liquid flow by adopting low speed and high pressure, and the liquid separation partition plate in the magnetic bead container is designed to effectively ensure the uniformity of the liquid flowing through the surface of the magnetic bead, so that the adsorption efficiency of the magnetic bead is maximized.
Advantageous effects
The invention organically combines tangential flow microfiltration, tangential flow ultrafiltration and magnetic bead adsorption, integrates a set of separation system, separates and purifies exosomes and active macromolecules in cell culture solution, realizes extraction strategies synchronously carried out by various principles, overcomes the defects of a single method, further strengthens the advantages of the single method, and greatly improves the purity and the extraction efficiency of products.
Drawings
FIG. 1 is a schematic diagram of the overall structure of a separation and purification device for stem cell exosomes and paracrine active macromolecules;
FIG. 2 is a schematic view of a hollow fiber microfiltration membrane;
FIG. 3 is a schematic view of a hollow fiber ultrafiltration membrane;
FIG. 4 is a front view, a side view, and a cross-sectional side view of a vessel containing low-adsorption magnetic beads;
FIG. 5 is a schematic view of the suction bucket;
FIG. 6 is a schematic diagram of the piping system for the isolation and purification of the exosomes of stem cell in example 2;
FIG. 7 is a flow chart of the isolation and purification of stem cell exosomes;
FIG. 8 is a schematic diagram of the piping system for isolating and purifying a paracrine active macromolecule of stem cells in example 3;
FIG. 9 is a flow chart of the isolation and purification of a stem cell active macromolecule for secretion;
FIG. 10 is a schematic diagram of a piping system for separating and purifying the exosomes of the stem cells in example 4;
FIG. 11 is a schematic diagram of a piping system for separating and purifying the exosomes of the stem cells in example 5;
the system comprises a touch operation screen 1, a filter fixing frame 2, a pretreatment pump 3, a circulating pump 4, a hollow fiber ultrafiltration membrane fixing frame 5, a magnetic plate 6, a magnetic bead container fixing clamp 7, an electric pressure regulator 8, a main pipeline electric liquid flow detection clamp 9, a loop electric liquid flow detection clamp 10, a product suction pipeline liquid flow detection clamp 11, a suction bucket lower edge fixing groove 12, a product suction bucket electric propeller 13, a product suction bucket upper edge fixing groove 13.1, an electric propulsion sliding rail 13.2, a raw liquid cylinder base 14, a filtrate cylinder base 15, a hanger cross bar 16, a hanger cross bar 16.1, a hanger upright column 17, a hanger pipeline electric clamp cross bar 18, a hanger pipeline electric clamp 18.1, a hanger pipeline base 19, a hollow fiber microfiltration membrane 20.1, a liquid inlet II, a liquid outlet II, a filtrate outlet 20.3 and a filtrate outlet II 20.4. 21 is a hollow fiber ultrafiltration membrane, 21.1 is a liquid inlet II, 21.2 is a liquid outlet II, 21.3 is a filtrate outlet III, 21.4 is a filtrate outlet IV, 22 is a low adsorption magnetic bead container, 22.1 is a container cover, 22.2 is a container cabin, 22.3 is a liquid inlet III, 22.4 is a liquid outlet III, 22.5 is a liquid separation partition plate, 22.6 is a liquid outlet screen, 23 is a suction barrel, 23.1 is a suction pipe, 23.2 is a piston push-pull rod, and 23.3 is a liquid suction port.
Detailed Description
The embodiments shown in the drawings of the present invention are merely representative examples of the present invention, and not all embodiments of the present invention are shown. Therefore, all other embodiments obtained by a person skilled in the art based on the embodiments of the present invention without making any creative efforts shall fall within the protection scope of the present invention.
The overall structure schematic diagram of the separation and purification device for the stem cell exosomes and paracrine active macromolecules is shown in figure 1 and comprises a main control panel device, a liquid storage device and a liquid storage hanging frame;
the main control panel device comprises a touch operation screen 1, a pretreatment device, an ultrafiltration purification device and a suction device, wherein the liquid storage device comprises a stock solution cylinder base 14 and a filtrate cylinder base 15 and is used for fixing a stock solution cylinder and a filtrate cylinder, and liquid level detectors are arranged in the stock solution cylinder and the filtrate cylinder; the liquid storage rack is in a shape like a Chinese character 'wang', the cross rods are respectively a rack cross rod 16, a rack pipeline electric clamping cross rod 18 and a rack base 19 from top to bottom, the upright rods are rack upright posts 17, 6 hooks 16.1 are mounted on the rack cross rod 16 and used for fixing buffer liquid and eluent, 6 rack pipeline electric clamps 18.1 are mounted on the rack pipeline electric clamping cross rod 18 and used for controlling the buffer liquid and the eluent to flow into the filtrate cylinder; the ultrafiltration purification devices are 3 groups and are connected in series or in parallel through pipelines when in use;
the pretreatment device comprises a filter fixing frame 2 and a pretreatment pump 3, wherein the filter fixing frame 2 is used for fixing the hollow fiber microfiltration membrane 20, a stock solution cylinder is connected with a liquid inlet I20.1 of the hollow fiber microfiltration membrane 20 through the pretreatment pump 3 by a pipeline, a liquid outlet I20.2 of the hollow fiber microfiltration membrane 20 is connected with the stock solution cylinder, and a filtrate port I20.3 of the hollow fiber microfiltration membrane 20 is connected with a filtrate cylinder by a pipeline;
the ultrafiltration adsorption device comprises a circulating pump 4, a hollow fiber ultrafiltration membrane fixing frame 5, a magnetic plate 6, a magnetic bead container fixing clamp 7 and a power pressure regulator 8, wherein the hollow fiber ultrafiltration membrane fixing frame 5 is used for fixing a hollow fiber ultrafiltration membrane 21, the magnetic plate 6 and the magnetic bead container fixing clamp 7 are used for fixing a low adsorption magnetic bead container 22, a filtrate cylinder is connected with the hollow fiber ultrafiltration membrane 21 through the circulating pump 4, the hollow fiber ultrafiltration membrane 21 is connected with the power pressure regulator 8 through a pipeline, the low adsorption magnetic bead container 22 is selectively connected between the hollow fiber ultrafiltration membrane 21 and the power pressure regulator 8, a main pipeline electric liquid flow detection clamp 9 is arranged on a main pipeline of the power pressure regulator 8 leading to a suction barrel 23 and the filtrate cylinder in the ultrafiltration purification device, a loop electric liquid flow detection clamp 10 is arranged on a loop pipeline between the ultrafiltration purification device and the filtrate cylinder, and a suction pipeline liquid flow detection clamp 11 is arranged on a pipeline between the ultrafiltration purification device and the suction barrel 23;
the suction device is composed of a lower edge fixing groove 12 of the suction barrel, an electric propeller 13 of the suction barrel, a lower edge fixing groove 13.1 of the suction barrel and an electric propulsion sliding rail 13.2 and is used for fixing the suction barrel 23, and the suction barrel 23 is connected with the filtrate cylinder and the separation and purification device through pipelines; a suction channel liquid flow detection clamp 11 is arranged on a pipeline between the ultrafiltration purification device and the suction barrel 23.
The schematic diagrams of the hollow fiber microfiltration membrane 20 and the hollow fiber ultrafiltration membrane 21 are respectively shown in fig. 2 and fig. 3, the hollow fiber microfiltration membrane 20 comprises a liquid inlet I20.1, a liquid outlet I20.2, a filtrate port I20.3 and a filtrate port II 20.4, the liquid inlet I20.1 is connected with the pretreatment pump 3 through a pipeline, the liquid outlet I20.2 is connected with a stock solution cylinder through a pipeline, and the filtrate port I20.3 is connected with a filtrate cylinder; the hollow fiber ultrafiltration membrane 21 comprises a liquid inlet II 21.1, a liquid outlet II 21.2, a filtrate outlet III 21.3 and a filtrate outlet IV 21.4, wherein the liquid inlet I21.1 is connected with the circulating pump 4 through a pipeline, the liquid outlet II 21.2 is selectively connected with the low-adsorption magnetic bead container 22 or the electric pressure regulator 8 through a pipeline, and the filtrate outlet III 21.3 is connected with the waste liquid cylinder through a pipeline.
A front view and a side sectional view of a low-adsorption magnetic bead container 22 are shown in fig. 4, a low-adsorption magnetic bead container 23 includes a container cover 22.1, a container bin 22.3, a liquid inlet iii 22.3, and a liquid outlet iii 22.4, the container bin 22.3 is pointed-bottom-shaped, so that magnetic beads can be conveniently sucked, a liquid separating partition plate 22.5 is arranged in the container bin 22.3, a liquid outlet screen 22.6 is arranged at the position of the liquid outlet iii 22.4, so that magnetic beads are prevented from being washed out, the liquid inlet iii 22.3 is selectively connected with a hollow fiber microfiltration membrane 20 through a pipeline, and the liquid outlet iii 22.4 is selectively connected with an electric pressure regulator 8 through a pipeline.
The structure of the suction barrel 23 is schematically shown in fig. 5, and comprises a suction pipe 23.1, a piston push-pull rod 23.2, liquid suction ports 23.3 and 23.4 which are the lower edge of the suction barrel, 23.5 which is the upper edge of the suction barrel, and the liquid suction port 23.3 which is connected with a filtrate cylinder and a separation and purification device through pipelines.
The above-mentioned connecting pipes in the present invention are all silica gel pipes.
The method for using the device for separating and purifying the stem cell exosomes and the paracrine active macromolecules in the invention is explained by combining the attached drawings and the specific embodiment.
Example 1
The composition of the complete medium was: the mesenchymal stem cells are free of serum basal medium and 5% serum substitute;
the basic culture medium comprises the following components: DMEM/F12+1.5% D-glucose + 50 μmol/L L-ascorbic acid;
specification of the hollow fiber membrane bioreactor: molecular weight cut-off 100kD (95% rejection), membrane surface area 1.2m 2 The volume of the external space of the hollow fiber is 70mL; pretreating the hollow fiber membrane bioreactor with PBS and a culture medium according to the specification;
(1) 2D culture of seed cells: separating Wharton jelly from fresh umbilical cord in sterile environment, suspending tissue blocks with complete culture medium, spreading in a bottle, observing cell growth under an inverted microscope, and digesting and passaging when 80% of cells are healed; 1X 10 in terms of cell density 4 /cm 2 Re-bottling, changing the liquid every 3 days, repeating the passage process when the cells grow to 80% union, and culturing at 37 deg.C、5%CO 2 ；
(2) 3D culture of a hollow fiber membrane bioreactor: taking P3 generation cells 5 x 10 8 Resuspending with serum-free complete medium, inoculating to hollow fiber membrane bioreactor external space, connecting hollow fiber internal space to complete medium storage tank, starting circulating pump with initial medium volume of 250mL, setting pump speed to medium speed, placing the device at 37 deg.C, and 5% CO 2 Culturing in an incubator, sampling from a culture medium storage tank every 12h, measuring the glucose content by using a glucose measuring instrument, replacing a new culture medium when the glucose is consumed by 80%, controlling the volume of the new culture medium to be gradually increased every time every 2 days according to the glucose consumption speed, and controlling the liquid to be replaced every day after 10 days;
(3) Culturing in a hollow fiber membrane bioreactor until day 10, wherein the culture medium consumption speed tends to be stable, beginning to harvest culture solution rich in exosomes from the external space of the hollow fiber every day, harvesting about 70mL each time, filling the external space of the hollow fiber with new complete culture medium, and beginning to obviously reduce the culture medium consumption speed about day 40, wherein the cell density of the external space of the hollow fiber is saturated, collecting the culture solution for the last time to obtain culture solution rich in stem cell exosomes, and filling the external space of the hollow fiber with new complete culture medium; separating the obtained culture solution rich in the exosome of the stem cells at low temperature (4 ℃,24 hours) and by centrifugation (1200 g, centrifugation for 10 min), removing cell debris and supernatant obtained after protein precipitation for later use;
(4) Harvesting of paracrine active macromolecules of stem cells: replacing the basic culture medium without biomacromolecules with the culture medium in the culture medium storage tank for culture; transferring the device to a three-gas incubator, adjusting the culture parameters to 37 ℃ and 5% CO 2 、5%O 2 (ii) a Collecting all old culture medium from the culture medium storage tank every 24h for extracting the paracrine bioactive macromolecules of the stem cells, equivalently replacing the basic culture medium, repeating the harvesting steps at 48h and 72h, finishing the cell culture process after 72h to obtain culture solution rich in the paracrine bioactive macromolecules of the stem cells, centrifuging (1200 g, centrifuging for 10 min) the obtained culture solution to remove the paracrine bioactive macromolecules of the stem cellsThe cell debris was used to obtain the supernatant.
Example 2
The schematic diagram of the pipeline system for separating and purifying the stem cell exosomes in example 2 is shown in fig. 6, the flowchart for separating and purifying the stem cell exosomes is shown in fig. 7, and the treatment capacity of each batch is 150mL;
the specific method for separating and purifying the stem cell exosomes comprises the following steps:
1) Installing and fixing a separation and purification device for a stem cell exosome and a paracrine active macromolecule, installing and fixing a stock solution cylinder, a filtrate cylinder, a hollow fiber micro-filtration membrane 20 (with the aperture of 0.22 mu m), a hollow fiber ultrafiltration membrane 21 (with the molecular weight cutoff of 100 kD), a low adsorption magnetic bead container 22 (filled with commercialized streptomycin affinity magnetic beads and a cross-linked biotin labeled Tim4 antibody inside) and a suction barrel 23, and connecting pipelines according to the figure 6, wherein the low adsorption magnetic bead container 22 is connected between the hollow fiber ultrafiltration membrane 21 and an electric pressure regulator 8, a culture solution rich in the stem cell exosome is placed in the stock solution cylinder, a buffer solution and an eluent are respectively fixed on a hook 16.1 arranged on a hanger cross rod 16, and the flow of the buffer solution and the eluent is respectively controlled by a hanger pipeline electric clamp 18.1;
2) Starting a pretreatment pump 3, setting the pump speed to be medium speed, filtering culture solution rich in stem cell exosomes in a stock solution cylinder by a hollow fiber microfiltration membrane 20, collecting all filtrate to a filtrate cylinder for filtration, closing the pretreatment pump 3 after the culture solution in the stock solution cylinder is exhausted, and entering the next step;
3) Automatically starting a circulating pump 4, setting the pump speed to be low, adjusting the pressure of a pressure regulator 8 to be high, performing circulating filtration until the volume of a filtrate is 1/10 of the original volume, suspending the circulating pump 4, opening an electric hanger pipeline clamp 18.1 corresponding to a buffer solution, allowing the buffer solution to flow into a filtrate cylinder, automatically closing the electric hanger pipeline clamp 18.1 when the volume of the filtrate in the filtrate cylinder reaches 150mL, starting the circulating pump 4, continuing the circulating filtration until the volume of the filtrate is 1/50 of the original volume of the culture solution, suspending the circulating pump 4, opening the electric hanger pipeline clamp 18.1 corresponding to the buffer solution, allowing the buffer solution to flow into the filtrate cylinder, automatically closing the electric hanger pipeline clamp 18.1 when the volume of the filtrate in the filtrate cylinder reaches 150mL, starting the circulating pump 4, continuing the circulating filtration until the volume of the filtrate is 1/50 of the original volume of the culture solution, suspending the circulating pump 4, and entering the next step;
4) Step 3) after ultrafiltration and affinity adsorption are finished, opening the electric hanger pipeline clamp 18.1 corresponding to the eluent, enabling the eluent to flow into a filtrate cylinder, automatically closing the electric hanger pipeline clamp 18.1 corresponding to the eluent when the volume of liquid in the filtrate cylinder is increased to 1/10, starting the circulating pump 4, completely opening the pressure regulator 8, circularly eluting for 60s, closing the circulating pump 4 after the elution is finished, and entering the next step;
5) The electric propeller 13 is started to suck the liquid in the filtrate cylinder and the pipeline system into the suction barrel 23, so as to complete the separation and purification of the exosome; when the main pipeline electric liquid flow detection clamp 9, the loop electric liquid flow detection clamp 10 and the suction pipeline liquid flow detection clamp 11 simultaneously pass through liquid continuously, the clamping function is activated and is in an open state; when no liquid flows through the pipeline, the clamps automatically clamp, when the three clamps clamp, the suction is automatically stopped, the separation and purification process of the stem cell exosomes is finished, and the stem cell exosomes are obtained in the suction barrel 23;
6) If the eluent needs to be replaced according to the application of the subsequent product, a dialysis bag can be used for buffer solution replacement; if sterilization is required, filtering and sterilizing by using a 0.22uM filter membrane;
7) After the separation procedure is completed for one time, starting an automatic cleaning procedure, flushing a pipeline system by using a buffer solution, and then carrying out next batch of separation; the hollow fiber membrane can be repeatedly used under the condition that the separation speed is not obviously reduced, and the immunoaffinity magnetic beads can be repeatedly used for 5 times.
Example 3
The schematic diagram of the pipeline system for separating and purifying the paracrine active macromolecules of the stem cells in example 2 is shown in fig. 8, the flow chart of the separation and purification of the exosomes of the stem cells is shown in fig. 9, and the treatment capacity of each batch is 150mL;
the specific method for separating and purifying the paracrine active macromolecules of the stem cells comprises the following steps:
1) Installing and fixing a separation and purification device for a stem cell exosome and a paracrine active macromolecule, installing and fixing a stock solution cylinder, a filtrate cylinder, a hollow fiber micro-filtration membrane 20 (with the aperture of 0.1 mu m), a hollow fiber ultrafiltration membrane 21 (with the throttling molecular weight of 5 kD) and a suction barrel 23, and connecting pipelines according to the structure shown in a figure 8, wherein the hollow fiber ultrafiltration membrane 21 is connected with an electric pressure regulator 8, a culture solution rich in the stem cell paracrine active macromolecule is placed in the stock solution cylinder, a buffer solution is fixed on a hook 16.1 arranged on a hanger cross rod 16, and the flowing-in of the buffer solution is controlled by a hanger pipeline electric clamp 18.1;
2) Starting a pretreatment pump 3, setting the pump speed to be medium speed, filtering a culture solution rich in the stem cell paracrine active macromolecules in a stock solution cylinder by a hollow fiber micro-filtration membrane 20, collecting all filtrate to a filtrate cylinder for filtration, closing the pretreatment pump 3 after the culture solution in the stock solution cylinder is exhausted, and entering the next step;
3) Automatically starting a circulating pump 4, setting the pump speed to be low, adjusting the pressure of a pressure regulator 8 to be high, performing circulating filtration until the volume of filtrate is 1/10 of the original volume, suspending the circulating pump 4, opening an electric hanger pipeline clamp 18.1 corresponding to a buffer solution, allowing the buffer solution to flow into a filtrate cylinder, automatically closing the electric hanger pipeline clamp 18.1 when the volume of filtrate in the filtrate cylinder reaches 150mL, starting the circulating pump 4, continuing the circulating filtration until the volume of filtrate is 1/50 of the volume of original culture solution, suspending the circulating pump 4, opening the electric hanger pipeline clamp 18.1 corresponding to the buffer solution, allowing the buffer solution to flow into the filtrate cylinder, automatically closing the electric hanger pipeline clamp 18.1 when the volume of filtrate in the filtrate cylinder reaches 150mL, starting the circulating pump 4, continuing the circulating pump to circulate until the volume of filtrate is 1/50 of the volume of original culture solution, suspending the circulating pump 4, and entering the next step;
4) The electric propeller 13 is started to suck the liquid in the filtrate cylinder and the pipeline system into the suction barrel 23, so as to complete the separation and purification of the paracrine active macromolecules; when the main pipeline electric liquid flow detection clamp 9, the loop electric liquid flow detection clamp 10 and the suction pipeline liquid flow detection clamp 11 simultaneously pass through liquid continuously, the clamping function is activated and is in an open state; when no liquid flows through the pipeline, the clamps automatically clamp, when the three clamps clamp, the suction is automatically stopped, the separation and purification process of the stem cell exosomes is finished, and a stem cell exosome product is obtained in the suction barrel 23;
5) According to the subsequent product application, if sterilization is required, filtering and sterilizing by using a 0.22uM filter membrane;
6) After the separation procedure is completed for one time, starting an automatic cleaning procedure, flushing a pipeline system by using a buffer solution, and then carrying out next batch of separation; the medium control fiber membrane can be recycled under the condition that the separation speed is not obviously reduced.
Example 4
Example 4 a schematic diagram of a piping system for separating and purifying exosomes of stem cells is shown in fig. 10, and three groups of ultrafiltration purification devices are connected in series; the rest of the operation was the same as in example 1.
Example 5
Example 5 a schematic diagram of a piping system for separating and purifying exosomes of stem cells is shown in fig. 11, three groups of ultrafiltration purification devices are connected in parallel; the rest of the operation was the same as in example 1.
Comparative example 1
The line system was connected according to FIG. 8, and the stem cell exosomes were extracted by ultrafiltration, and the other operations were the same as in example 2.
Data analysis
Total protein content was measured by BCA method, (50-150 nm) total number of particles was measured by NanoSight nanoparticle analyzer and representative biomacromolecule (VEGF, GDNF, bFGF) concentration was measured by ELISA method.
The total amount of protein, the total number of exosome particles and the separation and purification time before and after the stem cell exosomes are separated and purified in the examples 2, 4 to 5 and the comparative example 1 are detected and recorded, and the results are shown in the following table 1, and as can be seen from the table 1, the separation speed of the separation and purification method is obviously superior to that of the comparative example 1 (single hollow fiber ultrafiltration membrane treatment), and the protein recovery rate and the recovery rate of the exosome particles are also obviously superior to that of the comparative example 1.
The total protein content of the paracrine active macromolecules of the stem cells and the concentration of the representative biological macromolecules (VEGF, GDNF and bFGF) before and after the separation and purification of the example 3 are detected, and the results are shown in the following table 2.
TABLE 1 comparison of the results of the isolation and purification of exosomes from stem cells
TABLE 2 result of separation and purification of active macromolecules of exosomes from stem cells
Claims (8)
1. A separation and purification device for stem cell exosomes and paracrine active macromolecules is characterized by comprising a main control panel device, a liquid storage device and a liquid storage hanging rack;
the main control panel device comprises a touch operation screen (1), a pretreatment device, an ultrafiltration purification device and a suction device, wherein the liquid storage device comprises a stock solution cylinder base (14) and a filtrate cylinder base (15) and is used for fixing a stock solution cylinder and a filtrate cylinder, a liquid storage hanging rack is in a shape like a Chinese character 'wang', a hanging rack transverse rod (16), a hanging rack pipeline electric clamping transverse rod (18) and a hanging rack base (19) are respectively arranged on the transverse rod from top to bottom, the vertical rod is a hanging rack upright post (17), a plurality of hooks (16.1) are arranged on the hanging rack transverse rod (16) and are used for fixing buffer solution and eluent, a plurality of hanging rack pipeline electric clamps (18.1) are arranged on the hanging rack pipeline electric clamping transverse rod (18) and are used for controlling the inflow of the buffer solution and the eluent, and the buffer solution and the eluent are selectively connected with a filtrate cylinder pipeline;
the pretreatment device comprises a filter fixing frame (2) and a pretreatment pump (3), wherein the filter fixing frame (2) is used for fixing a hollow fiber microfiltration membrane (20), a stock solution cylinder is connected with the pretreatment pump (3) and the hollow fiber microfiltration membrane (20) through pipelines, the hollow fiber microfiltration membrane (20) is connected with the stock solution cylinder through a pipeline, and the hollow fiber microfiltration membrane (20) is connected with a filtrate cylinder through a pipeline and is used for collecting filtrate;
the ultrafiltration purification device comprises a circulating pump (4), a hollow fiber ultrafiltration membrane fixing frame (5), a magnetic plate (6), a magnetic bead container fixing clamp (7) and a power pressure regulator (8), wherein the hollow fiber ultrafiltration membrane fixing frame (5) is used for fixing a hollow fiber ultrafiltration membrane (21), the magnetic plate (6) and the magnetic bead container fixing clamp (7) are used for fixing a low-adsorption magnetic bead container (22), a filtrate cylinder is in pipeline connection with the circulating pump (4) and the hollow fiber ultrafiltration membrane (21), the hollow fiber ultrafiltration membrane (21) is in pipeline connection with the power pressure regulator (8), a selective pipeline between the hollow fiber ultrafiltration membrane (21) and the power pressure regulator (8) is in pipeline connection with the low-adsorption magnetic bead container (22), and the power pressure regulator (8) is in pipeline connection with the filtrate cylinder;
the suction device comprises a suction barrel, an electric propeller (13) of the suction barrel, a fixed groove (13.1) and an electric propulsion sliding rail (13.2) which are arranged below the suction barrel, and is used for fixing the suction barrel (23), wherein the suction barrel (23) is connected with a filtrate cylinder and an ultrafiltration purification device through pipelines.
2. The device for separating and purifying stem cell exosomes and paracrine active macromolecules according to claim 1, wherein the ultrafiltration purification devices are 1-3 groups and are connected in series or in parallel.
3. The device for separating and purifying stem cell exosomes and paracrine active macromolecules according to claim 1, wherein the hollow fiber microfiltration membrane (20) comprises a liquid inlet (20.1), a liquid outlet (20.2), a filtrate port I (20.3), a filtrate port II (20.4), and a filtrate port I (20.3) connected with a filtrate cylinder; the hollow fiber ultrafiltration membrane (21) comprises a liquid inlet II (21.1), a liquid outlet II (21.2), a filtrate port III (21.3) and a filtrate port IV (21.4); the low-adsorption magnetic bead container (22) comprises a container cover (22.1), a container bin (22.2), a liquid inlet III (22.3) and a liquid outlet III (22.4), wherein a liquid separation partition plate (22.5) is arranged in the container bin (22.2), and a liquid outlet screen (22.6) is arranged on the liquid outlet III (22.4); the suction barrel (23) comprises a suction pipe (23.1), a piston push-pull rod (23.2) and a liquid suction port (23.3).
4. The device for separating and purifying stem cell exosomes and paracrine active macromolecules according to claim 1, wherein a main pipeline electric liquid flow detection clamp (9) is arranged on a main pipeline of a power pressure regulator (8) in the ultrafiltration and purification device, which leads to a suction barrel (23) and a filtrate cylinder, a loop electric liquid flow detection clamp (10) is arranged on a loop pipeline between the ultrafiltration and purification device and the filtrate cylinder, and a suction loop liquid flow detection clamp (11) is arranged on a pipeline between the ultrafiltration and purification device and the suction barrel (23).
5. The apparatus for separating and purifying exosomes and paracrine active macromolecules of stem cells according to claim 1, wherein liquid level detectors are arranged in the stock solution tank and the filtrate tank.
6. The device for separating and purifying the stem cell exosomes and the paracrine active macromolecules of claim 1, wherein a pipeline for connecting the devices is a silica gel pipeline.
7. A method for using the device for separating and purifying the stem cell exosome and the paracrine active macromolecule according to any one of claims 1 to 6, which is characterized in that,
the method for separating and purifying the stem cell exosomes comprises the following steps:
1) installing and fixing a separation and purification device for stem cell exosomes and paracrine active macromolecules, installing and fixing a stock solution cylinder, a filtrate cylinder, a hollow fiber microfiltration membrane (20), a hollow fiber ultrafiltration membrane (21), a low adsorption magnetic bead container (22) and a suction barrel (23), communicating all pipelines, placing a culture solution rich in the stem cell exosomes in the stock solution cylinder, respectively fixing a buffer solution and an eluent on a hook (16.1), and respectively controlling the buffer solution and the eluent to flow into the filtrate cylinder by a rack pipeline electric clamp (18.1);
2) Starting a pretreatment pump (3), filtering culture solution rich in stem cell exosomes in a stock solution tank through a hollow fiber microfiltration membrane (20), and collecting filtrate into a filtrate tank;
3) Step 2) after the pretreatment, turning on a circulating pump (4) and adjusting the pressure of a pressure regulator (8) to ensure that the liquid in a filtrate cylinder is sequentially subjected to ultrafiltration and affinity adsorption through a hollow fiber ultrafiltration membrane (21) and a low adsorption magnetic bead container (22), feeding the treated filtrate into the filtrate cylinder, circularly performing ultrafiltration and affinity adsorption, and supplementing a buffer solution into the filtrate cylinder in the ultrafiltration and affinity adsorption processes by controlling an electric clamp (18.1) of a hanger pipeline corresponding to the buffer solution;
4) Step 3), after ultrafiltration and affinity adsorption treatment are finished, closing a circulating pump (4), opening a rack pipeline electric clamp (18.1) corresponding to the eluent, adding the eluent into a filtrate cylinder, closing the rack pipeline electric clamp (18.1), starting the circulating pump (4) for elution, eluting exosomes adsorbed on magnetic beads, and after elution is finished, closing the circulating pump (4);
5) After the elution is finished, the electric propeller (13) is started, and the liquid in the filtrate cylinder and the pipeline system is sucked into the suction barrel (23) to complete the separation and purification of the exosome;
the method for separating and purifying the paracrine active macromolecules comprises the following steps:
1) A stem cell exosome and paracrine active macromolecule separation and purification device is installed and fixed, a stock solution cylinder, a filtrate cylinder, a hollow fiber micro-filtration membrane (20), a hollow fiber ultrafiltration membrane (21) and a suction barrel (23) are installed and fixed, a culture solution rich in paracrine active macromolecules is placed in the stock solution cylinder, a buffer solution is fixed on a hook (16.1), and the buffer solution is controlled to flow into the filtrate cylinder by a hanger pipeline electric clamp (18.1);
2) Starting a pretreatment pump (3), filtering a paracrine active macromolecule-rich culture solution in a stock solution tank by a hollow fiber microfiltration membrane (20), and collecting filtrate to a filtrate tank;
3) Step 2) after the pretreatment is finished, opening a circulating pump (4) and adjusting the pressure of a pressure regulator (8) to ensure that the liquid in a filtrate cylinder is ultrafiltered by a hollow fiber ultrafiltration membrane (21), the treated filtrate enters the filtrate cylinder and is ultrafiltered circularly, and the buffer solution is supplemented into the filtrate cylinder in an ultrafiltration process by controlling an electric clamp (18.1) of a hanger pipeline corresponding to the buffer solution;
4) After the ultrafiltration is finished, the electric propeller (13) is started, and the liquid in the filtrate cylinder and the pipeline system is sucked into the suction barrel (23) to finish the separation and purification of paracrine active macromolecules.
8. The use method according to claim 7, wherein the magnetic beads in the low adsorption magnetic bead container (22) are streptomycin affinity magnetic beads and are cross-linked with a biotin-labeled Tim4 antibody.
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