A cell separation gel, preparation method and application thereof

Abstract

The present invention relates to a cell separation technology, and in particular to a cell separation gel, a preparation method and application thereof. The cell separation gel is a sodium alginate hydrogel cross-linked with calcium ions, wherein the mass concentration of sodium alginate in the cell separation gel is 7% to 9%, the calcium ions are evenly distributed inside the gel, and cross-linked with guluronic acid on the sodium alginate, and the cross-linking degree of the calcium ions with the sodium alginate is 0.3 to 0.5. The cell separation gel provided by the present invention can form a slit with the wall of a centrifuge tube to achieve separation of cell components, and can achieve separation at a lower centrifugal speed, which helps to maintain platelet activity.

Description

Technical Field

[0001] The present invention belongs to the field of tissue repair / regeneration, in particular to the repair / regeneration of skin, musculoskeletal system, oral and maxillofacial tissue, reproductive system, nervous system, and visual system, and specifically relates to a cell separation technology, namely a cell separation gel, and its preparation method and application. Background Art

[0002] Centrifuged platelet-rich plasma (PRP) is one of the important means of treatment in modern medicine; however, this route to PRP faces challenges, including the need for large amounts of blood collection, high centrifugation rates, long centrifugation cycles, and multiple centrifugations. These shortcomings bring inconvenience to patients' treatment, and more importantly, platelets are prone to lose activity after long periods of time and multiple centrifugation, thus missing the optimal treatment window.

[0003] Thixotropic hydrogel is a hydrogel with thixotropic behavior. Thixotropy is a fluid property that refers to the fact that some substances are gel-like or highly viscous when at rest, but become more fluid or less viscous when subjected to shear forces (such as stirring or vibration); when the shear force stops, the substance will gradually return to a gel-like or highly viscous state. Therefore, thixotropic hydrogels can maintain structural stability when at rest, and become soft and easy to flow under the action of external forces. Due to its thixotropy, PRP can be obtained by centrifuging whole blood once and for a short time. Unfortunately, the known thixotropic hydrogels are only used for in vitro medical testing (RAR Bowen, DM Adcock. Blood collection tubes as medical devices: The potential to affect assays and proposed verification and validation processes for the clinical laboratory, Clin. Biochem. 49 (18)(2016) 1321-1330, http: / / dx.doi.org / 10.1016 / j.clinbiochem.2016.10.004.), mainly because toxic components (such as glutaraldehyde) are used in the preparation process of the gel and cannot be used for in vivo therapeutic purposes (B.Ballantyne, SL Jordan. Toxicological, medical and industrial hygiene aspects of glutaraldehyde with particular reference to its biocidal use incold sterilization procedures, J. Appl.Toxicol.21(2)(2001)131-151, http: / / dx.doi.org / 10.1002 / jat.741.).Another problem with currently known thixotropic gels is that they cannot form a strong enough separation barrier between whole blood components due to their low viscosity recovery rate (the gel can still flow when the centrifuge is stopped), which may contaminate PRP (J. Emerson, S. Raghavan, K. Polymers for use in centrifugal separation of liquids [P]. United States Patent No. 9248447 B2, 2016.) or capture a large number of platelets (JKR Batra, N. Sadick, S. Batra. Where have all the platelets gone? A simple solution to the suboptimal performance of PRP tubes that contain a thixotropic gel separator, J. Cosmet. Dermatol. (2024), http: / / dx.doi.org / 10.1111 / jocd.16337.), resulting in separation failure. In addition, currently available thixotropic separation gels usually require density adjustment to 1.04 g / cm. 3 Near (between the density of whole blood red blood cells and the density of platelets / serum), for density separation or filtration separation; this separation principle is not applicable to patients whose plasma / serum density exceeds the standard level, limiting the indications for PRP treatment. Therefore, it is necessary to develop a safe gel to break through the thixotropic gel-assisted PRP separation principle.

[0004] The applicant's prior patent (patent number: CN2022115602785) discloses a gel separation method based on a horizontal centrifugal rotor. The sodium alginate gel used can only achieve platelet separation in a horizontal centrifuge. As shown in Comparative Example 4 in the patent, the gel cannot effectively achieve platelet separation in a fixed-angle rotor centrifuge. Since horizontal centrifuges have not yet been commercialized, it is necessary to develop a more ideal platelet separation technology based on the existing fixed-angle centrifuges with a high degree of commercialization. Summary of the invention

[0005] In view of the problem that the gel used in the existing fixed-angle centrifuge contains toxic components and cannot be used for in vivo treatment, the present invention proposes a PRP separation gel, which can achieve a small blood collection volume (3mL), one-step centrifugation, fast processing speed (3~5min), and low centrifugal speed (900~1500RPM), with little damage to platelets.

[0006] In order to achieve the above object, the present invention provides a cell separation gel in the first aspect:

[0007] The cell separation gel is a sodium alginate hydrogel cross-linked with calcium ions, wherein the mass concentration of sodium alginate in the cell separation gel is 7% to 9%, the calcium ions are evenly distributed inside the gel and cross-linked with the guluronic acid on the sodium alginate, and the cross-linking degree between the calcium ions and the sodium alginate is 0.3 to 0.5, preferably 0.4.

[0008] Preferably, the calcium ions are cross-linked with the guluronic acid on the sodium alginate by ionic bonds.

[0009] Preferably, the density of the cell separation gel is 1.1 g / cm 3 .

[0010] Preferably, the molar ratio of guluronic acid to mannuronic acid in the sodium alginate is 1:2.

[0011] In a second aspect, the present invention provides a method for preparing a cell separation gel, the method comprising: cross-linking sodium alginate with calcium ions to form a sodium alginate hydrogel cross-linked with calcium ions; wherein the mass concentration of the sodium alginate is 7% to 9%.

[0012] Preferably, the step of cross-linking sodium alginate and calcium ions is as follows: adding sodium alginate to a sodium citrate solution, dissolving it, and then adding a calcium source to disperse it in the solution, wherein the calcium source is a compound with low solubility in water, and continuous stirring can form a suspension, and the calcium source is evenly dispersed in the solution; adding an acid to release calcium ions from the suspended calcium source to obtain a calcium-cross-linked sodium alginate gel; since the calcium source is evenly dispersed in the solution, after adding the acid, the released calcium ions are also evenly distributed in the solution, and a sodium alginate hydrogel with calcium ions evenly distributed inside the gel can be formed.

[0013] Preferably, in order to further improve the uniformity of the gel, the calcium source is continuously stirred at 90-100° C. to form a uniformly dispersed suspension. Heating at 90-100° C. not only helps to uniformly disperse the calcium source in the formed suspension, but also inhibits the reaction between calcium and sodium alginate, that is, slows down the cross-linking reaction rate, and can more finely control the uniformity of the gel, which further contributes to controlling the uniformity of the gel.

[0014] Preferably, the compound with low solubility in water is selected from at least one of calcium carbonate, calcium phosphate, calcium oxalate, calcium sulfate, calcium hydrogen phosphate, calcium fluoride, calcium selenate, calcium silicate and the like, which is poorly soluble in water but soluble in acid. The acid is selected from at least one of citric acid, hydrochloric acid and sulfuric acid, which can dissolve the calcium source compound and release calcium ions to cross-link with sodium alginate; citric acid is preferred because it is considered to be a relatively safe acid in the medical field.

[0015] Preferably, the molar ratio of guluronic acid to mannuronic acid in the sodium alginate is 1:2.

[0016] Preferably, the mass concentration of the sodium citrate in the solution is 4%.

[0017] Preferably, the mass concentration of the sodium alginate in the solution is 7% to 9%.

[0018] Preferably, the mass concentration of the calcium source in the solution is 0.37-0.62%.

[0019] Preferably, the mass concentration of the acid in the solution is 0.47%-0.79%.

[0020] The above method can be used to prepare a gel with a cross-linking degree of 0.3-0.5. Calcium ions and guluronic acid form ionic bonds, forming an "egg-box" model as a whole: the structure becomes loose when sheared, and the structure recovers when the shearing stops, so it has thixotropy.

[0021] The third aspect of the present invention provides a use of the cell separation gel in the preparation of PRP.

[0022] Preferably, the application comprises the following steps:

[0023] (1) Place the cell separation gel at the bottom of a centrifuge tube, and then add anticoagulated whole blood;

[0024] (2) Place the above centrifuge tube in an angle centrifuge for centrifugation. The supernatant after centrifugation is the PRP without red blood cells.

[0025] Preferably, in the angle centrifugation, the angle between the centrifuge tube and the rotating shaft is 45°.

[0026] Preferably, the centrifugal speed is 900-1500 revolutions per minute (RPM).

[0027] Preferably, the centrifugation time is 3 to 5 minutes.

[0028] Sodium alginate is a byproduct of extracting iodine and mannitol from brown algae such as kelp or sargassum. Its molecule is composed of β-D-mannuronic acid (β-D-mannuronic, M) and α-L-guluronic acid (α-L-guluronic, G) connected by (1→4) bonds. In the present invention, the formation of sodium alginate hydrogel is due to Ca 2+ It interacts with G residues to form a chain bond, thus forming a connecting region. Therefore, the cross-linking degree between sodium alginate and calcium is Φ = Ca 2+ / G, that is, the cross-linking degree is the mass fraction of calcium ions in sodium alginate / the mass fraction of guluronic acid. By adjusting the amount of calcium ions and guluronic acid, the cross-linking degree of the gel can be controlled to be 0.3~0.5.

[0029] The cell separation gel provided by the present invention has the following main characteristics:

[0030] 1. Sodium alginate and calcium are cross-linked to obtain thixotropic properties that can be quickly recovered, thereby achieving angle centrifuge separation; therefore, the separation gel of the present invention is different from the reported separation gel in separation mechanism. The present invention uses the slit formed between the gel and the centrifuge tube wall to achieve separation, while the existing gels all use the gel to block platelets to achieve separation (red blood cells pass through the gel, and platelets also have the risk of being embedded in the gel). The "slit" separation described in the present invention utilizes the "local" shear thinning of the thixotropic gel. Under the action of centrifugal force, the weak ionic bonds between the calcium ions and the sodium alginate G residues will be destroyed, and the interaction force between the calcium ions and the sodium alginate G residues will be reduced, resulting in the gel structure becoming loose and the gel viscosity decreasing. Therefore, under the action of centrifugal force, the cross-section of the gel in the vertical direction will be deformed, the volume of the gel will shrink, and the distance between the gel and the tube wall will widen to form a gap; at the same time, the blood cells move toward the tube wall under the action of centrifugal force, and move toward the bottom of the tube through the slit under the action of gravity. Since the mass of platelets is less than that of red blood cells and white blood cells, the sedimentation rate of platelets will be less than that of red blood cells and white blood cells. Therefore, mainly red blood cells and white blood cells settle to the bottom of the gel through the narrow gap, while platelets mainly remain on the upper part of the gel, thereby achieving blood cell separation. When the centrifugal speed gradually decreases, according to the thixotropy of the gel, during the process of stopping shearing, the viscosity of the gel will be restored, the volume of the gel will be restored, the gaps will disappear, and finally the gel will form a soft barrier between the blood components to achieve platelet enrichment.

[0031] 2. The components of the separation gel of the present invention are highly safe and can be used for PRP separation for therapeutic purposes (existing gels have toxicity risks).

[0032] 3. The existing gel needs to adjust its density to between the density of red blood cells and platelets (+ serum) (usually 1.04 g / cm 3 ), when the platelet (+ serum) density of the patient changes slightly, separation cannot be achieved; however, the present invention does not require strict control of the gel density between the separated cells (the gel density in this case is 1.1 g / cm 3 , and effective separation can also be achieved).

[0033] 4. The platelet concentration after centrifugation using the separation gel of the present invention is 1.4 to 2.1 times that of the original whole blood. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the separation result of Example 1 of the present invention;

[0035] Figure 2 This is the infrared spectrum of the sodium alginate calcium ion gel prepared in Example 2 of the present invention;

[0036] Figure 3 This is the separation result of Example 2 of the present invention;

[0037] Figure 4 This is the separation result of Example 3 of the present invention;

[0038] Figure 5 This is the separation result of Example 4 of the present invention;

[0039] Figure 6 This is the separation result of Example 5 of the present invention;

[0040] Figure 7 This is the separation result of Example 6 of the present invention;

[0041] Figure 8 This is the separation result of Example 7 of the present invention;

[0042] Fig. 9 This is the separation result of Example 8 of the present invention;

[0043] Fig.10 This is the separation result of Comparative Example 1. DETAILED DESCRIPTION

[0044] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0045] Sodium alginate (SA, analytical grade, 90%, M / G=2:1), citric acid (99.5%) and calcium carbonate (99%) were purchased from Sanofi Chemical Reagent Co., Ltd. The centrifuge model used in the embodiment of the present invention is JOANLAB LC-6S PRP. Whole blood was anticoagulated with sodium citrate.

[0046] Unless otherwise specified in the present invention, the percentages are all by mass.

[0047] Example 1

[0048] The total mass of the gel was maintained at 100 g, which was the sum of the masses of the components, namely sodium alginate, sodium citrate, calcium carbonate, citric acid, and deionized water (the same applies to the following embodiments).

[0049] 4 g of anhydrous sodium citrate was added to 88.02 g of deionized water to prepare a sodium citrate solution, and then 7 g of sodium alginate was added to the sodium citrate solution, and the mixture was heated to 96° C. to completely dissolve the sodium alginate to prepare a sodium alginate solution; after stirring for 45 minutes, 0.43 g of calcium carbonate was added to the sodium alginate solution; the mixture was stirred at 96° C. for 1 hour to form a uniformly dispersed suspension, and 0.55 g of citric acid was added to release calcium ions in the suspended calcium carbonate, thereby forming a calcium-crosslinked sodium alginate calcium ion gel with a crosslinking degree of 0.4 and a density of 1.1 g / cm 3 .

[0050] Take 2mL of the prepared gel and place it at the bottom of a 15mL centrifuge tube, then add 3mL of anticoagulated whole blood; centrifuge the tube containing sodium alginate calcium ion gel and whole blood in a centrifuge with a fixed angle (45°) rotor at 1100 revolutions per minute (RPM) for 4 minutes; the gel can effectively separate red blood cells and platelets ( Figure 1 ), the platelet concentration in the prepared PRP is 2.1 times that of the original whole blood.

[0051] Example 2

[0052] 4 g of anhydrous sodium citrate was added to 86.87 g of deionized water to prepare a sodium citrate solution, and then 8 g of sodium alginate was added to the sodium citrate solution, and the mixture was heated to 96° C. to completely dissolve the sodium alginate to prepare a sodium alginate solution; after stirring for 45 minutes, 0.49 g of calcium carbonate was added to the sodium alginate solution; the mixture was stirred at 96° C. for 1 hour to form a uniformly dispersed suspension, and 0.64 g of citric acid was added to release calcium ions in the suspended calcium carbonate, thereby forming a calcium-crosslinked sodium alginate calcium ion gel with a crosslinking degree of 0.4 and a density of 1.1 g / cm 3 .

[0053] The gel was dried at 50 °C for 2 h to prepare a film. The Fourier transform infrared (FTIR) spectra of the film were recorded using a Nicolet is50 (Thermo Fisher Scientific (China) Co., Ltd.) in situ infrared spectrometer in attenuated total reflection mode (ATR). Each spectrum represents the average of 32 scans ranging from 700 to 4000 cm -1 , resolution 4cm -1 The results are as follows Figure 2 As shown, the sample group without calcium does not add calcium source and citric acid; other addition amounts and processes are the same as those in this embodiment.

[0054] like Figure 2 As shown, the infrared spectrum is 946.83 cm -1 and 892.40 cm -1Corresponding to the mannuronic acid unit. There is no shift in the two absorption peaks of the samples with calcium addition (8% SAΦ0.4) and without calcium addition (8% SA), indicating that calcium is not bound to the mannuronic acid unit; 1587.66 cm -1 and 1404.77 cm -1 These peaks were shifted (to 1577.39 cm -1 and 1396.71 cm -1 ), indicating that it is combined with calcium. It shows that the method of the present invention can prepare a sodium alginate gel containing calcium ion cross-linking.

[0055] Take 2mL of the prepared gel and place it at the bottom of a 15mL centrifuge tube, then add 3mL of anticoagulated whole blood; centrifuge the tube containing sodium alginate gel and whole blood in a centrifuge with a fixed angle (45°) rotor at 1100 revolutions per minute (RPM) for 4 minutes; the gel can effectively separate red blood cells and platelets ( Figure 3 ), the platelet concentration in the prepared PRP is 1.8 times that of the original whole blood.

[0056] Example 3

[0057] 4 g of anhydrous sodium citrate was added to 85.73 g of deionized water to prepare a sodium citrate solution, 9 g of sodium alginate was added to the sodium citrate solution, and the mixture was heated to 96° C. to completely dissolve the sodium alginate to prepare a sodium alginate solution; after stirring for 45 minutes, 0.55 g of calcium carbonate was added to the sodium alginate solution; the mixture was stirred at 96° C. for 1 hour to form a uniformly dispersed suspension, and 0.72 g of citric acid was added to release calcium ions in the suspended calcium carbonate, thereby forming a calcium-crosslinked sodium alginate calcium ion gel with a crosslinking degree of 0.4 and a density of 1.1 g / cm 3 .

[0058] Take 2mL of the prepared gel and place it at the bottom of a 15mL centrifuge tube, then add 3mL of anticoagulated whole blood; centrifuge the tube containing sodium alginate gel and whole blood in a centrifuge with a fixed angle (45°) rotor at 1100 revolutions per minute (RPM) for 4 minutes; the gel can effectively separate red blood cells and platelets ( Figure 4 ), the platelet concentration in the prepared PRP is 1.8 times that of the original whole blood.

[0059] Example 4

[0060] 4 g of anhydrous sodium citrate was added to 87.16 g of deionized water to prepare a sodium citrate solution, 8 g of sodium alginate was added to the sodium citrate solution, and the mixture was heated to 96° C. to completely dissolve the sodium alginate to prepare a sodium alginate solution; after stirring for 45 minutes, 0.37 g of calcium carbonate was added to the sodium alginate solution; the mixture was stirred at 96° C. for 1 hour to form a uniformly dispersed suspension, and 0.47 g of citric acid was added to release calcium ions in the suspended calcium carbonate, thereby forming a calcium-crosslinked sodium alginate calcium ion gel with a crosslinking degree of 0.3 and a density of 1.1 g / cm 3 .

[0061] Take 2mL of the prepared gel and place it at the bottom of a 15mL centrifuge tube, then add 3mL of anticoagulated whole blood; centrifuge the tube containing sodium alginate gel and whole blood in a centrifuge with a fixed angle (45°) rotor at 1100 revolutions per minute (RPM) for 4 minutes; the gel can effectively separate red blood cells and platelets ( Figure 5 ), the platelet concentration in the prepared PRP is 1.9 times that of the original whole blood.

[0062] Example 5

[0063] 4 g of anhydrous sodium citrate was added to 86.59 g of deionized water to prepare a sodium citrate solution, 8 g of sodium alginate was added to the sodium citrate solution, and the mixture was heated to 96° C. to completely dissolve the sodium alginate to prepare a sodium alginate solution; after stirring for 45 minutes, 0.62 g of calcium carbonate was added to the sodium alginate solution; the mixture was stirred at 96° C. for 1 hour to form a uniformly dispersed suspension, and 0.79 g of citric acid was added to release calcium ions in the suspended calcium carbonate, thereby forming a calcium-crosslinked sodium alginate calcium ion gel with a crosslinking degree of 0.5 and a density of 1.1 g / cm 3 .

[0064] Take 2mL of the prepared gel and place it at the bottom of a 15mL centrifuge tube, then add 3mL of anticoagulated whole blood; centrifuge the tube containing sodium alginate gel and whole blood in a centrifuge with a fixed angle (45°) rotor at 1100 revolutions per minute (RPM) for 4 minutes; the gel can effectively separate red blood cells and platelets ( Figure 6 ), the platelet concentration in the prepared PRP is 1.8 times that of the original whole blood.

[0065] Embodiment 6-8

[0066] The same sodium alginate calcium ion gel as in Example 2 was used, except that the centrifugal speed was 900 RPM, 1300 RPM and 1500 RPM. The gel can effectively separate red blood cells and platelets ( Figure 7-9), the platelet concentrations in the prepared PRP were 1.6 times, 1.7 times and 1.4 times that of the original whole blood respectively.

[0067] Comparative Example 1

[0068] The cell separation gel used is the same as that in Example 2, except that the separation is performed by horizontal centrifugation. The horizontal centrifugation method used can refer to the horizontal centrifugation device in Chinese patent CN2022115602785. The horizontal centrifugation results are shown in FIG. Fig.10 As shown. Fig.10 It can be seen that at 1100RPM and below, the gel cannot achieve cell separation; at 1300RPM and above, separation is possible, but the red blood cells are mixed in the gel and PRP through the gel (it can be seen that both the gel and PRP have been dyed red more evenly).

Claims

1. Application of a cell separation gel in the preparation of PRP, characterized in that: The cell separation gel is a sodium alginate hydrogel cross-linked with calcium ions, wherein the mass concentration of sodium alginate in the cell separation gel is 7% to 9%, the calcium ions are evenly distributed inside the gel and cross-linked with the guluronic acid on the sodium alginate, and the cross-linking degree between the calcium ions and the sodium alginate is 0.3 to 0.

5.

2. The use of the cell separation gel according to claim 1 in the preparation of PRP, characterized in that: The cross-linking degree between the calcium ions and the sodium alginate is 0.

4.

3. Use of the cell separation gel according to claim 1 in the preparation of PRP, characterized in that: The density of the cell separation gel is 1.1 g / cm 3 .

4. Use of the cell separation gel according to claim 1 in the preparation of PRP, characterized in that: The molar ratio of guluronic acid to mannuronic acid in the sodium alginate is 1:

2.

5. Use of the cell separation gel according to any one of claims 1 to 4 in the preparation of PRP, characterized in that: The cell separation gel is prepared by the following method: sodium alginate is added to a sodium citrate solution and dissolved, a calcium source is added and dispersed in the solution, and stirring is continued to form a uniformly dispersed suspension; acid is added to the solution to make the calcium source release calcium ions to obtain a calcium-crosslinked sodium alginate gel.

6. Use of the cell separation gel according to claim 5 in the preparation of PRP, characterized in that: The calcium source is continuously stirred at 90-100° C. to form a uniformly dispersed suspension.

7. Use of the cell separation gel according to claim 5 in the preparation of PRP, characterized in that: The calcium source is a compound with low solubility in water, selected from at least one of calcium carbonate, calcium phosphate, calcium oxalate, calcium sulfate, calcium hydrogen phosphate, calcium fluoride, calcium selenate, and calcium silicate; the acid is selected from at least one of citric acid, hydrochloric acid, and sulfuric acid.

8. Use of the cell separation gel according to claim 5 in the preparation of PRP, characterized in that: The mass concentration of the sodium citrate in the solution is 4%; the mass concentration of the sodium alginate in the solution is 7%-9%; the mass concentration of the calcium source in the solution is 0.37-0.62%; and the mass concentration of the acid in the solution is 0.47%-0.79%.

9. Use of the cell separation gel according to claim 1 in the preparation of PRP, characterized in that: The application comprises the following steps: (1) Place the cell separation gel at the bottom of a centrifuge tube, and then add anticoagulated whole blood; (2) Place the above centrifuge tube in an angle centrifuge and centrifuge. The supernatant is the PRP without red blood cells.

10. The use according to claim 9, characterized in that In the angle centrifugation, the angle between the centrifuge tube and the rotating shaft is 45°; the centrifugal speed is 900-1500 revolutions per minute; and the centrifugal time is 3-5 minutes.

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