Microcarrier freeze-dried bead and preparation method thereof

Abstract

The invention provides a microcarrier freeze-dried bead and a preparation method thereof. The microcarrier freeze-dried beads are prepared by the following steps: uniformly mixing a wet microcarrier with an excipient, dispersing and dripping the mixture into liquid nitrogen, quickly freezing into small solid round ice balls with uniform and regular forms, and then freeze-drying to form the freeze-dried beads. The freeze-dried bead is uniform in particle size and round and full in shape, one freeze-dried bead contains 150-4000 microcarriers, the freeze-dried bead has a loose net structure and is quick to redissolve, and the redissolved microcarriers are good in dispersity. The form of the microcarrier freeze-dried beads changes the conventional form of freeze-dried powder of the microcarrier, and basically eliminates the electrostatic adsorption phenomenon of the microcarrier freeze-dried powder, and solves the problems that the microcarrier freeze-dried powder is difficult to weigh, easy to fly and pollute, difficult to subpackage and the like. A certain amount of microcarriers are contained in a single freeze-dried bead, so that quantitative use is facilitated. A freeze-dried bead skeleton structure formed by the excipient formula has dispersion and protection effects on the microcarrier, so that the freeze-dried beads cannot be separated during transportation, are quickly redissolved and dispersed during use, and can be subsequently used for cell culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterials and cell culture technology, and particularly relates to a kind of microcarrier freeze-dried beads and a preparation method thereof. BACKGROUND

[0002] Microcarriers have been widely used in medicine, cosmetics, biomaterials and cell culture technology due to their excellent biocompatibility, biodegradability and high drug loading capacity. In cell culture, compared with traditional two-dimensional culture and other three-dimensional techniques (such as hydrogel embedding, hanging drop method, etc.), microspheres with high specific surface area not only can construct three-dimensional structure scaffold, but also can highly simulate in vivo microenvironment, providing a highly biomimetic, controllable and scalable culture condition for cells, thereby effectively overcoming the space and functional limitations of two-dimensional culture. Microcarriers are not only a physical scaffold to support cell "three-dimensional growth", but also a micro-bioreactor with rich functions and precise control. Their high-density culture characteristics are conducive to realizing large-scale and automated operation, significantly promoting the collaborative development of life science frontier exploration and biopharmaceutical industry.

[0003] Microcarriers are key materials for large-scale culture of adherent-dependent cells, usually microspheres with a diameter of 100-300 microns, providing a surface for cell attachment growth. In order to be stored for a long time, microcarriers usually exist in the form of liquid dispersion system (suspended in liquid) or freeze-dried powder. However, the existing technology has the following significant defects: (1) Liquid dispersion system storage: high transportation cost (cold chain required), large space occupation, risk of contamination and leakage, and short shelf life. (2) Freeze-dried powder form: This is the most commonly used dry storage form, but it has inherent and difficult to overcome disadvantages: ① Static electricity problem: Dry microcarrier powder is prone to static electricity, causing it to adhere to the container wall and weighing spoon, resulting in inaccurate weighing and large material loss; ②Flying and pollution: Microcarrier powder is light, and it is easy to fly during opening, weighing and dispensing, not only causing waste, but also possibly contaminating the sterile operation table and cell culture environment, posing a serious threat to sterile operation; ③Quantitative difficulty: Due to static electricity and flying, it is difficult to accurately weigh a small amount (such as a few milligrams) of microcarriers, which brings difficulties to the repeatability of experiments and the standardization of large-scale production; ④Reconstitution agglomeration: Freeze-dried powder is prone to form agglomerates that are difficult to disperse during reconstitution due to surface hydrophobicity or static electricity, affecting the uniformity of subsequent cell inoculation.

[0004] 201910079680.3 discloses a cell carrier particle aggregate and a preparation method thereof, the aggregate morphology is sheet and block, and the cell carrier particles are punched and aggregated into a shape under the action of an external force by using a punching die. The method needs to use a punching die and punch the microcarriers under the action of an external force, which is easy to cause the rupture of the microcarriers and is not conducive to subsequent cell culture. Moreover, the production efficiency of the method is low, and the method cannot meet the needs of large-scale production.

[0005] Therefore, there is an urgent need in the art for a new microcarrier preparation form which can not damage the microcarriers, can be mass-produced and enlarged, and can avoid electrostatic adsorption and weighing loss. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide a microcarrier freeze-dried bead and a preparation method thereof. The microcarrier freeze-dried bead is convenient to store, transport and dose. The microcarrier freeze-dried bead can solve the problems of electrostatic adsorption, weighing difficulty, easy scattering pollution and easy aggregation during reconstitution of the microcarrier freeze-dried powder. The preparation method of the microcarrier freeze-dried bead is simple in process and good in reproducibility, and is suitable for large-scale production.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a preparation method of a microcarrier freeze-dried bead, which comprises:

[0009] (1) Preparation of a suspension: mixing wet microcarriers and excipients in a liquid medium to form a microcarrier suspension;

[0010] (2) Drop freezing molding: using a drop device to drop the suspension into liquid nitrogen drop by drop to obtain ice balls;

[0011] (3) Collection and freeze-drying: collecting the ice balls in liquid nitrogen and transferring them to a pre-cooled freeze-drying machine to perform main drying and desorption drying to remove excess water and form microcarrier freeze-dried beads maintaining a spherical structure.

[0012] In the present application, wet microcarriers and specific excipients are prepared into a suspension according to a certain proportion, the suspension is added dropwise into liquid nitrogen through a drop device, the extremely low temperature (-196℃) of the liquid nitrogen causes the droplets to freeze instantly, forming solid spherical ice crystal particles with regular shape and uniform size, the microcarriers and excipients are uniformly fixed in the ice crystal framework, the ice balls are intercepted and collected by a screen, and the excess liquid nitrogen is removed. The collected frozen particles are then subjected to freeze-drying under low-temperature vacuum conditions to remove water, and finally the microcarrier freeze-dried beads with loose internal structure and certain mechanical strength are obtained.

[0013] The microcarrier freeze-dried bead form changes the conventional freeze-dried powder form of the microcarrier, substantially eliminates the common electrostatic adsorption phenomenon of the microcarrier freeze-dried powder, and solves the problems of difficulty in weighing, easy scattering and pollution, and difficulty in sub-packaging during use of the microcarrier freeze-dried powder. The single freeze-dried bead contains a certain amount of microcarriers, which is convenient for quantitative use, avoids the inaccuracy of weighing of the freeze-dried powder microcarriers due to static electricity, and the dispersed microcarriers after reconstitution can maintain the cell culture performance of the microcarriers.

[0014] Preferably, in step (1), the microcarriers are selected from any one or a combination of at least two of natural polymer microspheres, synthetic polymer microspheres, or inorganic material microspheres.

[0015] Preferably, the natural polymer microspheres are selected from any one or a combination of at least two of gelatin microspheres, collagen microspheres, chitosan microspheres, dextran microspheres, alginate microspheres, starch microspheres, or albumin microspheres.

[0016] Preferably, the synthetic polymer microspheres are selected from any one or a combination of at least two of PLGA microspheres, PLA microspheres, PCL microspheres, or PGA microspheres.

[0017] Preferably, the inorganic material microspheres are selected from any one or a combination of at least two of silica microspheres, magnetic microspheres, or hydroxyapatite microspheres.

[0018] In a preferred embodiment, the microcarriers are 3D StarPore microcarriers.

[0019] Preferably, in step (1), the liquid medium is selected from any one or a combination of at least two of water, phosphate buffer, citrate buffer, acetate buffer, borate buffer, or Tris buffer.

[0020] In the present application, the liquid medium serves as the dispersion phase of the microspheres and the excipient.

[0021] Preferably, in step (1), the mass ratio of the wet-state microcarriers to the excipient is 1:(0.1-20), for example, it can be 1:0.1, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:18, or 1:20, etc.

[0022] Preferably, the mass ratio of the wet-state microcarriers to the excipient is 1:(1-20), preferably 1:(5-20), and further preferably 1:(6-20).

[0023] In the present application, too many microspheres and too little excipient, the excipient does not play a role in excipient; too little microspheres and too much excipient, the whole lyophilized beads will be more dense, the rehydration is not so good, but also lost the meaning of lyophilized beads itself, as a microsphere aggregate, in a lyophilized bead will gather a large number of microspheres.

[0024] Preferably, in step (1), the excipient is selected from any one or a combination of at least two of sucrose, mannitol, sorbitol, trehalose, lactose, galactose, glycine, arginine, leucine, histidine, proline, methionine, glucose, cyclodextrin, collagen, human serum albumin, bovine serum albumin, casein, dextran, hydroxyethyl starch, polyvinylpyrrolidone, polyethylene glycol, chitosan, hyaluronic acid, gelatin and its derivatives, polysorbate 20, polysorbate 80, sodium chloride, potassium chloride, calcium chloride, phosphate, citrate, acetate, borate or Tris salt.

[0025] In the present application, if only simple microspheres are used for infusion, after lyophilization, it is still one by one small microspheres, not large lyophilized beads, through the adhesion, filling, disintegration and dispersion of the excipient, the microspheres can form large lyophilized beads, without affecting the morphology, stability, usability and rehydration of the microspheres.

[0026] The lyophilized bead skeleton structure formed by the excipient formula of the present application plays a dispersing and effective protection role on the microcarriers, so that the lyophilized beads do not separate during transportation, and can be quickly reconstituted and dispersed during use. The microcarrier lyophilized beads of the present application can be further sterilized by irradiation for storage, which is convenient for direct use in subsequent cell culture, can be stored and transported at room temperature, and greatly reduces the transportation cost.

[0027] Preferably, in step (2), the droplet device is selected from any one of a syringe, a dropper, a droplet machine, a precision pipette, a precision injection pump, a liquid separation system or a liquid handling workstation.

[0028] Preferably, the diameter of the ice ball is 1-20 mm, for example, it can be 1 mm, 5 mm, 10 mm, 15 mm or 20 mm, etc.

[0029] Preferably, in step (3), the pre-cooling temperature of the freeze dryer is -(40±1)℃~-(50±1)℃.

[0030] In the present application, the ice ball is low temperature, if the freeze dryer is not pre-cooled, the ice ball is put into the room temperature, during the operation of the freeze dryer, the ice ball will melt, the morphology of the microspheres will also be affected during the freeze drying process, and then the use will be affected.

[0031] In the present application, if only the excipient is simply added and direct ly freeze-dried, the freeze-dried product is a block of freeze-dried powder, rather than a large freeze-dried bead. The block of freeze-dried powder needs to be shaken hard to be dispersed into powder, the dry powder shows strong electrostatic adsorption when being weighed, and the dry powder needs to be shaken hard to be dispersed when being reconstituted, and there are still a small amount of small clumps. Microscope observation shows that the microcarriers after reconstitution are severely broken and the morphology is destroyed, which affects the morphology, stability, usability and rehydration of the microspheres.

[0032] In the present application, if no excipient is added and only liquid nitrogen infusion and freeze-drying are simply performed, the freeze-dried product is a dispersed dry powder, that is, a dispersed microsphere dry powder, rather than a large freeze-dried bead with clear particles. Although some irregular hemispheres are composed of a plurality of microspheres gathered together, they can be dispersed into uniform microsphere particles by gently touching. When being weighed, the dry powder shows strong electrostatic adsorption. Microscope observation shows that the morphology does not change after reconstitution, and the properties of the dry powder are the same as those of the microsphere dry powder.

[0033] In a second aspect, the present application provides a microcarrier freeze-dried bead prepared by the preparation method of the microcarrier freeze-dried bead according to the first aspect.

[0034] Preferably, the microcarrier freeze-dried bead is a regular three-dimensional spherical structure or a multi-layer spherical structure; the particle size of the microcarrier freeze-dried bead ranges from 2 mm to 10 mm (for example, it can be 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm, etc.); the inside of the microcarrier freeze-dried bead contains 150-4000 (for example, it can be 150, 200, 500, 1000, 2000, 3000 or 4000, etc.) independent microcarriers, which are embedded and uniformly dispersed in the loose porous reticular framework structure formed by the excipient.

[0035] In the present application, the regular three-dimensional spherical structure is formed by embedding and uniformly dispersing the microcarriers in the loose porous reticular framework structure formed by the excipient.

[0036] In the present application, the regular multi-layer spherical structure is formed by embedding and uniformly dispersing the microcarriers in the loose porous reticular framework structure formed by the excipient according to a certain direction.

[0037] In the present application, the microcarrier freeze-dried bead has uniform morphology and loose reticular structure, and can quickly release the microcarriers after reconstitution; one freeze-dried bead contains 150-4000 microcarriers, which can be directly used quantitatively without weighing; the electrostatic adsorption is low and easy to sub-pack; the freeze-dried bead can be directly used for cell culture after irradiation; it can be stored and transported at room temperature, and the microcarrier cell adsorption performance after reconstitution is not lower than that of the dry powder freeze-dried microcarrier.

[0038] In the present application, the electrostatic adsorption strength of the microcarrier freeze-dried beads is reduced by more than 80% than that of the conventional freeze-dried microcarrier powder, and there is no flying phenomenon in the A-level laminar flow environment.

[0039] In the present application, the obtained freeze-dried beads are subjected to irradiation sterilization (such as gamma-ray or electron beam irradiation), and then vacuum or nitrogen filling packaging is performed, so that normal temperature storage and transportation can be realized.

[0040] In the third aspect, the present application provides the application of the microcarrier freeze-dried beads of the second aspect, which can realize quantitative dispensing of the microcarriers by counting the particles, and the whole or multiple particles can be directly taken for use.

[0041] In the present application, the microcarrier freeze-dried beads realize quantitative dispensing of the microcarriers by counting the particles, and the whole or multiple particles can be directly taken for use each time, so that repeated weighing pollution is avoided.

[0042] The numerical range in the present application not only includes the point values listed above, but also includes any point values between the above numerical ranges which are not listed, and the specific point values included in the range are not listed due to the length and the consideration of simplicity.

[0043] Compared with the prior art, the present application has the following beneficial effects:

[0044] (1) The present application changes the microcarriers from amorphous "powder" form to regular and discrete "bead" solid form for the first time. This fundamental change in form is the physical basis for overcoming all the disadvantages of freeze-dried powder.

[0045] (2) The present application creates a unique "bead-in-bead" structure through a specific excipient formula and liquid nitrogen drop freezing process. The porous framework formed by the excipient not only protects the microcarriers from dissociation during freeze-drying and transportation, but also ensures that water can quickly penetrate during reconstitution, so that the microcarriers are quickly and uniformly dispersed.

[0046] (3) The freeze-dried beads of the present application completely solve the problems of static electricity and flying, and the freeze-dried beads are large in size and heavy in quality, and basically do not produce electrostatic adsorption and will not fly during operation, greatly improving the operation safety and convenience and avoiding cross contamination.

[0047] (4) The use of the freeze-dried beads can realize accurate quantification: a single freeze-dried bead contains a certain amount of microcarriers (such as 150-4000 per bead), and the user can directly count ("how many beads") instead of the cumbersome and inaccurate weighing. This greatly simplifies the operation process, improves the repeatability of the experiment and the standardization level of the production process.

[0048] (5) The freeze-dried beads have excellent reconstitution performance: the loose reticular structure ensures that the freeze-dried beads can be quickly reconstituted when contacting with culture medium or buffer, the internal microcarriers can be well dispersed without aggregation, and excellent cell culture performance is maintained.

[0049] (6) The freeze-dried beads are easy to package, store and transport: the bead shape is easy to be divided into standard containers such as a flask. The product can be stored and transported at room temperature after irradiation sterilization, completely eliminating the dependence on cold chain, and significantly reducing the cost and risk.

[0050] (7) The preparation process of the freeze-dried beads is controllable and easy to scale up: the liquid nitrogen drop freezing method is easy to control the particle size of the freeze-dried beads, and the process reproducibility is good, which is very suitable for scale-up production from laboratory to industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 is a process flow chart of the preparation method of the microcarrier freeze-dried beads.

[0052] Figure 2 is a macroscopic morphology diagram of the microcarrier freeze-dried beads.

[0053] Figure 3 is a macroscopic morphology diagram of the freeze-dried beads prepared by different types and proportions of excipients.

[0054] Figure 4 is a macroscopic morphology diagram of the sample of Example 1 and Comparative Example 1 before and after reconstitution.

[0055] Figure 5 is a macroscopic morphology diagram of the sample of Comparative Example 2.

[0056] Figure 6 is a diagram of the state of the microcarrier freeze-dried beads before and after reconstitution and dispersion during reconstitution.

[0057] Figure 7 is a diagram of the state of the microcarrier freeze-dried beads before and after reconstitution and dispersion during reconstitution.

[0058] Figure 8 is a diagram of the state of the microcarrier freeze-dried beads before and after reconstitution and dispersion during reconstitution. DETAILED DESCRIPTION

[0059] The technical solutions of the present application will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present application and should not be regarded as specific limitations of the present application.

[0060] If a specific technology or condition is not specified in the embodiments, it is performed according to the technology or condition described in the literature in the art or according to the product instruction. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be commercially available through a regular channel.

[0061] Example 1

[0062] This embodiment provides a method for preparing microcarrier freeze-dried beads. Figure 1 This is a process flow diagram for the preparation of freeze-dried beads of microcarriers.

[0063] The preparation method of microcarrier freeze-dried beads includes the following steps:

[0064] (1) Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, hydrate overnight at room temperature, wash with deionized water, centrifuge to remove supernatant, and obtain wet 3D StarPore microcarrier.

[0065] (2) Prepare an aqueous solution of excipients containing 50 mg / mL trehalose and 20 mg / mL mannitol.

[0066] (3) Mix the wet microcarrier with 10 mL of excipient solution and gently vortex to form a uniform suspension.

[0067] (4) Use a 1 mL syringe to draw up the suspension and drop it into the liquid nitrogen at a rate of 1 drop per second from a height of about 10 cm above the surface of the liquid nitrogen. Immediately, an ice ball with a diameter of about 4 mm is formed.

[0068] (5) Use a pre-cooled sieve to scoop out the ice balls, collect them and quickly transfer them to a freeze dryer pre-cooled at -40℃.

[0069] (6) Start the freeze-drying program: maintain at -40℃ for 2 hours, then increase the temperature to -10℃ at 0.5℃ / min and maintain for 5 hours; then increase the temperature to 25℃ at 0.3℃ / min and maintain for 8 hours. Maintain the vacuum degree at 10-30 Pa.

[0070] (7) After freeze-drying, white microcarrier freeze-dried beads were obtained, with the appearance as shown in the attached figure. Figure 2 As shown in the figure. According to the count, each freeze-dried bead contains an average of about 600 microcarriers.

[0071] In this embodiment, if only the mass ratio of wet microspheres to excipients or only the proportion of excipients is changed while other conditions remain constant, when the 3D StarPore microcarrier is fixed at 100 mg and the volume of the excipient solution is fixed at 10 mL, the concentrations of trehalose and mannitol are: 1 mg / mL trehalose, 10 mg / mL trehalose, 50 mg / mL trehalose, and 50 mg / mL trehalose + 10 mg / mL mannitol, respectively. The morphology of the lyophilized beads is as follows: Figure 3 As shown, from left to right, it gradually transforms into a smooth, compact, and regular three-dimensional sphere. From Figure 3It can be seen that when the proportion of excipients is low, the microspheres are difficult to aggregate, and it is difficult to form tight and smooth freeze-dried beads. With the increase of the proportion of excipients, the morphology of freeze-dried beads is better and better.

[0072] Comparative Example 1

[0073] The present comparative example provides a preparation method of microcarrier freeze-dried beads, which comprises the following steps:

[0074] (1) Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, hydrate overnight at room temperature, wash with deionized water, and then centrifuge to remove the supernatant to obtain wet 3D StarPore microcarriers.

[0075] (2) Prepare an aqueous excipient solution containing 50 mg / mL trehalose and 20 mg / mL mannitol.

[0076] (3) Mix the wet microcarriers with 10 mL of the excipient solution, and gently vortex to form a uniform suspension.

[0077] (4) Load the suspension into a vial for freeze-drying to obtain a freeze-dried powder; the freeze-drying program is as follows: -40°C for 2 hours, then 0.5°C / min to -10°C, 5 hours; then 0.3°C / min to 25°C, 8 hours. The vacuum degree is maintained at 10-30 Pa.

[0078] (5) After freeze-drying, the freeze-dried powder obtained in the present comparative example is in block shape, and the appearance is as shown in Figure 4 , which needs to be shaken by an instrument to disperse into powder, and the dry powder shows strong electrostatic adsorption during weighing, and needs to be vortexed violently to disperse during reconstitution, and there are still a small amount of small clumps. Microscopic observation found that the reconstituted microcarriers were severely broken and the morphology was destroyed.

[0079] Comparative Example 2

[0080] The present comparative example provides a preparation method of microcarrier freeze-dried beads, which comprises the following steps:

[0081] (1) Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, hydrate overnight at room temperature, wash with deionized water, and then centrifuge to remove the supernatant to obtain wet 3D StarPore microcarriers.

[0082] (2) Mix the wet microcarriers with 10 mL of water for injection, and gently vortex to form a uniform suspension.

[0083] (3) Take 1 mL of the suspension with a 1 mL syringe and drop into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a rate of 1 drop per second, and immediately form ice balls with a diameter of about 4 mm.

[0084] (4) Take out the ice balls with a pre-cooled mesh, collect and quickly transfer to a pre-cooled freeze dryer at -40°C.

[0085] (5) Start the freeze drying program: keep at -40°C for 2 hours, then increase the temperature to -10°C at a rate of 0.5°C / min, keep for 5 hours; then increase the temperature to 25°C at a rate of 0.3°C / min, keep for 8 hours. The vacuum degree is maintained at 10-30 Pa.

[0086] (6) After the freeze drying is completed, the comparative example 2 obtains dispersed and granular microspheres, and the appearance is as shown in Figure 5 Although some irregular hemispheres are formed by the aggregation of multiple microspheres according to the appearance, they can be dispersed into uniform microsphere particles by a slight touch, and exhibit strong electrostatic adsorption during weighing. Microscopic observation after reconstitution shows that the morphology does not change, and the properties are the same as those of the microsphere dry powder.

[0087] Test Example 1

[0088] The performance of the microcarrier freeze-dried beads is tested in this test example.

[0089] 1. Reconstitution test: take 5 freeze-dried beads prepared in Example 1 and put them into 10 mL of PBS buffer. The freeze-dried beads quickly absorb water and rapidly disintegrate, completely dissolving within 15 seconds, and the internal microcarriers are uniformly dispersed in the solution without visible clumps. The whole process is as shown in Figure 6 Microscopic observation is performed on the sample, and it can be seen from Figure 7 that the morphology of the microcarriers does not change before and after the formation of the freeze-dried beads, indicating that the structure of the freeze-dried beads is a good microcarrier aggregate. Figure 4 The state diagrams of the samples before and after reconstitution of Example 1 and Comparative Example 1 are shown in the figures. It can be seen from the figures that the microspheres of Example 1 completely dissolve within 15 seconds, and the internal microcarriers are uniformly dispersed in the solution without visible clumps, achieving rapid rehydration. The microspheres of Comparative Example 1 cannot completely dissolve and disperse in a short time, and a large number of clumps can be clearly seen at the bottom of the vial. Figure 5 The appearance diagram of the freeze-dried Comparative Example 2 is shown in Figure 2 and Figure 5 It can be seen that by adding the excipient and bonding, the microspheres can form large freeze-dried beads after liquid nitrogen drop freezing. Without the excipient, the microspheres are still small and granular after liquid nitrogen drop freezing, and it is difficult to form regular and compact freeze-dried beads with a three-dimensional spherical structure.

[0090] 2. Cell culture test: The sample of Example 1 was reconstituted by hydration with culture medium, and the reconstituted microcarriers were inoculated with human mesenchymal stem cells (hMSCs) for comparison with the microcarriers reconstituted from the lyophilized powder. As shown in FIG. 2, the cells on the microcarriers of the present application and the lyophilized powder were cultured for 72 h and 96 h, respectively. Figure 8 As can be seen, the cells on the dry powder group proliferated by 13.7 times after 72 h of culture, and the cells on the lyophilized bead group proliferated by 12.5 times. After 96 h of culture, the cells on the dry powder group proliferated by 17.8 times, and the cells on the lyophilized bead group proliferated by 17.4 times. The results show that there is no significant difference in cell adhesion rate, proliferation rate and final cell density between the two groups, which proves that the lyophilized bead process of the present application does not impair the biological function of the microcarriers.

[0091] 3. Quantitative convenience test: 10 mg of microcarriers (the lyophilized powder was weighed, and the lyophilized beads were counted) were weighed by an analytical balance and a counting method, respectively. The operation was repeated 10 times. The results show that the weighing error of the lyophilized powder group is as high as ± 15% due to electrostatic adsorption and scattering. The actual error of the lyophilized bead group is less than ± 5% by counting, and the operation time is shortened by 70%.

[0092] Example 2

[0093] The present example provides a preparation method of a microcarrier lyophilized bead, which is different from Example 1 only in that the excipient is mannitol and dextran, and the excipient solution contains 30 mg / mL mannitol and 10 mg / mL dextran.

[0094] 1. Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, and hydrate overnight at room temperature. After washing with deionized water, centrifuge to remove the supernatant, and obtain wet 3D StarPore microcarriers.

[0095] 2. Prepare an excipient aqueous solution containing 30 mg / mL mannitol and 10 mg / mL dextran.

[0096] 3. Mix the wet microcarriers with 15 mL of the excipient solution, and gently vortex to form a uniform suspension.

[0097] 4. Use a pipette to draw the suspension, and drop it into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a speed of 1 drop per second, to immediately form a white ice ball with a diameter of about 6 mm.

[0098] 5. Use a pre-cooled sieve to fish out the ice ball, and quickly transfer it to a pre-cooled freeze dryer at -40°C.

[0099] 6. Start the freeze-drying program: keep at -40°C for 2 hours, then increase the temperature to -10°C at a rate of 0.5°C / min, keep for 5 hours; then increase the temperature to 25°C at a rate of 0.3°C / min, keep for 8 hours. The vacuum degree is maintained at 10-30 Pa.

[0100] 7. After the end of freeze-drying, white microcarrier freeze-dried beads were obtained. Counting and statistics showed that each freeze-dried bead contained about 1400 microcarriers on average.

[0101] Example 3

[0102] This example provides a preparation method of microcarrier freeze-dried beads, which is different from Example 1 only in that the excipients are sucrose and dextran, and the aqueous excipient solution contains 100 mg / mL sucrose and 20 mg / mL dextran.

[0103] 1. 200 mg of 3D StarPore microcarrier dry powder was taken and added to 50 mL of deionized water. After hydration at room temperature overnight, the wet 3D StarPore microcarriers were obtained by washing with deionized water and centrifugation to remove the supernatant.

[0104] 2. An aqueous excipient solution containing 100 mg / mL sucrose and 20 mg / mL dextran was prepared.

[0105] 3. The wet microcarriers were mixed with 20 mL of the excipient solution, and a uniform suspension was formed by gentle vortexing.

[0106] 4. The suspension was taken with a 2 mL syringe and dropped into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a speed of 1 drop per second, immediately forming white ice balls with a diameter of about 5 mm.

[0107] 5. The ice balls were fished out with a pre-cooled sieve and quickly transferred to a pre-cooled freeze dryer at -40°C.

[0108] 6. The freeze-drying program was started: -40°C for 2 hours, then warmed to -10°C at a rate of 0.5°C / min, and kept for 5 hours; then warmed to 25°C at a rate of 0.3°C / min, and kept for 8 hours. The vacuum degree was maintained at 10-30 Pa.

[0109] 7. After the end of freeze-drying, white microcarrier freeze-dried beads were obtained. Counting and statistics showed that each freeze-dried bead contained about 1400 microcarriers on average.

[0110] Example 4

[0111] This example provides a preparation method of microcarrier freeze-dried beads, which is different from Example 1 only in that the excipients are glycine and mannitol, and the aqueous excipient solution contains 20 mg / mL glycine and 50 mg / mL mannitol.

[0112] 1. Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, hydrate at room temperature overnight, wash with deionized water, and centrifuge to remove supernatant to obtain wet 3D StarPore microcarriers.

[0113] 2. Prepare an excipient aqueous solution containing 20 mg / mL glycine and 50 mg / mL mannitol.

[0114] 3. Mix the wet microcarriers with 10 mL of the excipient solution, gently vortex to form a uniform suspension.

[0115] 4. Use a 1 mL syringe to draw the suspension, and drop it into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a rate of 1 drop per second, immediately forming a white ice ball with a diameter of about 4 mm.

[0116] 5. Use a pre-cooled mesh to fish out the ice ball and quickly transfer it to a pre-cooled freeze dryer at -40°C.

[0117] 6. Start the freeze-drying program: maintain at -40°C for 2 hours, then increase the temperature to -10°C at a rate of 0.5°C / min, maintain for 5 hours; then increase the temperature to 25°C at a rate of 0.3°C / min, maintain for 8 hours. The vacuum degree is maintained at 10-30 Pa.

[0118] 7. After the freeze-drying is completed, white microcarrier freeze-dried beads are obtained. Counting statistics show that each freeze-dried bead contains about 600 microcarriers on average.

[0119] Example 5

[0120] This example provides a method for preparing microcarrier freeze-dried beads, which is different from Example 1 only in that the excipients are dextran and leucine, and the excipient aqueous solution contains 10 mg / mL dextran and 20 mg / mL leucine.

[0121] 1. Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, hydrate at room temperature overnight, wash with deionized water, and centrifuge to remove supernatant to obtain wet 3D StarPore microcarriers.

[0122] 2. Prepare an excipient aqueous solution containing 10 mg / mL dextran and 20 mg / mL leucine.

[0123] 3. Mix the wet microcarriers with 10 mL of the excipient solution, gently vortex to form a uniform suspension.

[0124] 4. Take 1 mL of the suspension with a 1 mL syringe and drop into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a rate of 1 drop per second, which immediately forms a white ice ball with a diameter of about 3 mm.

[0125] 5. Retrieve the ice ball with a pre-cooled mesh screen and quickly transfer to a pre-cooled freeze dryer at -40 °C.

[0126] 6. Start the freeze-drying program: maintain at -40 °C for 2 hours, then increase the temperature to -10 °C at a rate of 0.5 °C / min, maintain for 5 hours; then increase the temperature to 25 °C at a rate of 0.3 °C / min, maintain for 8 hours. The vacuum level is maintained at 10-30 Pa.

[0127] 7. After the freeze-drying is completed, the white microcarrier lyophilized beads are obtained. Counting and statistics show that each lyophilized bead contains about 250 microcarriers on average.

[0128] Example 6

[0129] This example provides a method for preparing microcarrier lyophilized beads, which is different from Example 1 only in that the excipients are sodium chloride, dextran and mannitol, and the aqueous excipient solution contains 10 mg / mL sodium chloride, 20 mg / mL dextran and 20 mg / mL mannitol.

[0130] 1. Take 100 mg of 3D StarPore microcarrier dry powder, add 50 mL of deionized water, hydrate at room temperature overnight, wash with deionized water, then centrifuge to remove the supernatant, and obtain wet 3D StarPore microcarriers.

[0131] 2. Prepare an aqueous excipient solution containing 10 mg / mL sodium chloride, 20 mg / mL dextran and 20 mg / mL mannitol.

[0132] 3. Mix the wet microcarriers with 15 mL of the excipient solution, and gently vortex to form a uniform suspension.

[0133] 4. Take 1 mL of the suspension with a 1 mL syringe and drop into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a rate of 1 drop per second, which immediately forms a white ice ball with a diameter of about 3 mm.

[0134] 5. Retrieve the ice ball with a pre-cooled mesh screen and quickly transfer to a pre-cooled freeze dryer at -40 °C.

[0135] 6. Start the freeze-drying program: maintain at -40 °C for 2 hours, then increase the temperature to -10 °C at a rate of 0.5 °C / min, maintain for 5 hours; then increase the temperature to 25 °C at a rate of 0.3 °C / min, maintain for 8 hours. The vacuum level is maintained at 10-30 Pa.

[0136] 7. After the end of freeze-drying, the white microcarrier freeze-dried beads were obtained. Counting and statistics showed that each freeze-dried bead contained about 200 microcarriers on average.

[0137] Example 7

[0138] This example provides a preparation method of microcarrier freeze-dried beads, which is only different from Example 1 in that the excipient is human serum albumin and dextran, and the aqueous solution of the excipient contains 100 mg / mL human serum albumin and 10 mg / mL dextran.

[0139] 1. 200 mg of 3D StarPore microcarrier dry powder was taken and added to 50 mL of deionized water. After hydration at room temperature overnight, the wet 3D StarPore microcarriers were obtained by washing with deionized water and centrifugation to remove the supernatant.

[0140] 2. An aqueous solution of the excipient containing 100 mg / mL human serum albumin and 10 mg / mL dextran was prepared.

[0141] 3. The wet microcarriers were mixed with 25 mL of the excipient solution, and a uniform suspension was formed by gentle vortexing.

[0142] 4. The suspension was taken with a 2 mL syringe and dropped into liquid nitrogen at a height of about 10 cm from the liquid nitrogen surface at a rate of 1 drop per second, immediately forming white ice balls with a diameter of about 5 mm.

[0143] 5. The ice balls were fished out with a pre-cooled sieve and quickly transferred to a pre-cooled freeze dryer at -40°C.

[0144] 6. The freeze-drying program was started: -40°C for 2 hours, then warmed to -10°C at a rate of 0.5°C / min, and kept for 5 hours; then warmed to 25°C at a rate of 0.3°C / min, and kept for 8 hours. The vacuum degree was maintained at 10-30 Pa.

[0145] 7. After the end of freeze-drying, the white microcarrier freeze-dried beads were obtained. Counting and statistics showed that each freeze-dried bead contained about 1000 microcarriers on average.

[0146] Table 1 shows the test results of the microcarrier freeze-dried beads prepared in Examples 1-7 and Comparative Examples 1 and 2.

[0147] Table 1

[0148]

[0149] From the results of Table 1, it can be seen that the freeze-dried beads prepared in Examples 1-7 can be quickly rehydrated to form uniformly dispersed single microspheres, and the cell proliferation effect is not much different from that of the dry powder microspheres. In the weighing test, due to the low electrostatic adsorption of the freeze-dried beads, the particles are distinct and easy to count, so the operation time is short, and the weighing error is less than ± 5%. The sample in Comparative Example 1 is difficult to quickly rehydrate in a short time, and needs to be vortexed with great force during rehydration, and the internal microsphere structure is damaged. In cell culture, the cells only proliferated 5.8 times in 96 h, and the effect is not ideal. In addition, the sample in Comparative Example 2 has good cell culture effect, but it is difficult to form regular three-dimensional spherical freeze-dried beads.

[0150] From the comparison between Example 1 and Comparative Example 1, it can be seen that the freeze-dried beads prepared by liquid nitrogen dripping method have not been affected in physical properties and effectiveness, which shows that the freeze-dried beads are a good aggregation form of microspheres.

[0151] From the comparison between Example 1 and Comparative Example 2, it can be seen that only by adding excipients can smooth and regular three-dimensional spherical freeze-dried beads be prepared, and the excipients play an important role in the preparation of freeze-dried beads.

[0152] In summary, the microcarrier freeze-dried beads and the preparation method thereof provided by the present application successfully solve the long-standing technical problems in the field, and provide a new product form which is significantly superior to the traditional freeze-dried powder in terms of quantitative accuracy, operational convenience, storage and transportation economy and safety, and has very high industrial application value.

[0153] The applicant declares that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A method of preparing a microcarrier lyophilized bead, characterized in that, The preparation method comprises: (1) Suspension preparation: mixing wet microcarriers and excipients in a liquid medium to form a microcarrier suspension; (2) Liquid nitrogen drop freezing molding: using a drop device to drop the suspension into liquid nitrogen drop by drop to obtain ice balls; (3) Collection and freeze-drying: collecting the ice balls in liquid nitrogen and transferring them to a pre-cooled freeze dryer for main drying and analytical drying to remove excess water and form microcarrier freeze-dried beads maintaining spherical structure.

2. The method for preparing microcarrier freeze-dried beads according to claim 1, characterized in that, In step (1), the microcarriers are selected from any one or a combination of at least two of natural polymer microspheres, synthetic polymer microspheres or inorganic material microspheres.

3. The method of claim 2, wherein the microcarrier lyophilized beads are prepared by, The natural polymer microspheres are selected from any one or a combination of at least two of gelatin microspheres, collagen microspheres, chitosan microspheres, dextran microspheres, alginate microspheres, starch microspheres or albumin microspheres; Preferably, the synthetic polymer microspheres are selected from any one or a combination of at least two of PLGA microspheres, PLA microspheres, PCL microspheres or PGA microspheres; Preferably, the inorganic material microspheres are selected from any one or a combination of at least two of silica microspheres, magnetic microspheres or hydroxyapatite microspheres.

4. The method of preparing microcarrier lyophilized beads according to any one of claims 1-3, characterized in that, In step (1), the liquid medium is selected from any one or a combination of at least two of water, phosphate buffer, citrate buffer, acetate buffer, borate buffer or Tris buffer.

5. The method of preparing microcarrier lyophilized beads according to any one of claims 1-4, characterized in that, In step (1), the mass ratio of the wet microcarriers to the excipients is 1:(0.1-20).

6. The method of preparing microcarrier lyophilized beads according to any one of claims 1-5, wherein, In step (1), the excipients are selected from any one or a combination of at least two of sucrose, mannitol, sorbitol, trehalose, lactose, galactose, glycine, arginine, leucine, histidine, proline, methionine, glucose, cyclodextrin, collagen, human serum albumin, bovine serum albumin, casein, dextran, hydroxyethyl starch, polyvinylpyrrolidone, polyethylene glycol, chitosan, hyaluronic acid, gelatin and its derivatives, polysorbate 20, polysorbate 80, sodium chloride, potassium chloride, calcium chloride, phosphate, citrate, acetate, borate or Tris salt.

7. The method of preparing microcarrier lyophilized beads according to any one of claims 1-6, wherein, In step (2), the drop device is selected from any one of a syringe, a dropper, a droplet machine, a precision pipette, a precision syringe pump, a liquid distribution system or a liquid handling workstation; Preferably, the diameter of the ice balls is 1-20 mm.

8. A microcarrier lyophilized bead characterized in that, The microcarrier freeze-dried beads are prepared by the preparation method of the microcarrier freeze-dried beads according to any one of claims 1-7.

9. The microcarrier lyophilized bead according to claim 8, wherein, The microcarrier freeze-dried beads are regular three-dimensional spherical or multi-layer spherical structures; the particle size of the microcarrier freeze-dried beads ranges from 2 to 10 mm; the interior of the microcarrier freeze-dried beads contains 150-4000 independent microcarriers embedded and uniformly dispersed in a loose and porous reticular framework structure formed by the excipients.

10. Use of the microcarrier lyophilized beads according to claim 8 or 9, characterized in that, The microcarriers can be quantitatively dispensed by counting the particles, and the whole or multiple particles are taken for use.

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