CN116998458A - Animal model construction method of central nervous immune cell preparation - Google Patents
Animal model construction method of central nervous immune cell preparation Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
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- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
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Abstract
The application provides a method for constructing an animal model of a central nervous immune cell preparation, which comprises the following steps: s1, selecting a target animal and homologous close relatives of the target animal; s2, performing immune cell separation, reconstruction, amplification and purification on the homologous close parents of the target animal according to the operation specification of immune cell therapy to obtain an immune cell preparation from the homologous close parents; s3, injecting the syngeneic immune cell preparation into cerebrospinal fluid of the target animal by a intrathecal injection mode to obtain an animal model of the central nervous immune cell preparation. According to the animal model construction method, immune cells required by separation and purification of similar closely related mice are adopted, and after transformation, the immune cells are directly injected into cerebrospinal fluid by bypassing a blood brain barrier in an intrathecal injection mode, so that possible rejection reaction can be avoided in a short period, the requirements of animal experiment stages are met, and a favorable foundation is provided for developing research of central system diseases in the future.
Description
Technical Field
The application relates to the technical field of biological medicine, in particular to the technical field of animal model construction.
Background
Immune cell therapy is a treatment modality that kills tumors by reinfusion of immune cells of the patient itself or of the donor. Currently, the therapeutic schemes of Polyclone Treg, CAR-Treg, TCR-Treg, TIL, TCR-T (TCR-engineered T) cells and CAR-T cells are mainly used, and occupy most of the layout of the immune cell therapeutic market. The CAR-T cell therapy has great potential and application value in malignant blood tumors, TIL and TCR-T cell therapy have certain effects in application in a small part of solid tumors, but the effective response rate and beneficiaries of the immune cell therapy in most solid tumors are still limited, and the reasons thereof can be mainly summarized as factors such as antigen heterogeneity and immune escape, poor immune cell infiltration capability, immunosuppression and metabolic microenvironment obstruction of tumor microenvironment, T cell exhaustion and the like. For example, CAR-T cell therapy, chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy), is a novel immunotherapy that uses specifically transformed T cells to more specifically target cancer cells. The doctor first extracts a sample of T cells from the patient's blood and then reconstitutes these T cells, thereby allowing them to develop specific structures on their surface known as Chimeric Antigen Receptors (CARs). When these CAR-T cells are re-injected into a patient, these receptors can help T cells recognize, attack cancer cells in the human body.
Because of the existence of the blood brain barrier, macromolecular drugs and cells are difficult to enter brain tissues, and effective substances are difficult to reach the onset complement for treating central degenerative diseases, central autoimmune diseases and central system tumors, the clinical curative effect of related immune cell therapies is lost or hardly has curative effect.
Not only are clinical trials, autologous infusion of immune cell therapies, but also present a significant challenge to rodent experimental models, such as the mouse NOG model, various transgenic models, EAE model, cuprazone-induced mouse brain tissue demyelination model, SOD1 gene mutant mouse model, and the like. Because immune cell therapies directed against the central system are first obtained from autologous immune cells, and after the cells are prepared from the same mouse, the mouse is difficult to survive or withstand subsequent testing. In immune system sound mouse pathological models (EAE, demyelination model and SOD1 model as described above), cells prepared from syngeneic mice can only meet short-term pharmacodynamic test requirements if allogeneic vein or abdominal cavity or subcutaneous injection is adopted, and the problem of foreign body rejection can be presented through repeated administration. If immunodeficient mice, such as NOG mice, are used, the pharmacodynamic performance of immunosuppressive cells is disturbed. Meanwhile, due to the existence of a blood brain barrier, the cell preparation cannot enter the central system and reach the effective part. Thus, to date, no precedent has been seen regarding successful administration of cell therapy formulations to construct a mouse model.
Disclosure of Invention
Aiming at the problems, the application provides an animal model construction method of a central nervous immune cell preparation, which adopts immune cells needed by separation and purification of homologous mice, and directly injects cerebrospinal fluid by bypassing a blood brain barrier in a intrathecal injection mode after transformation, so that possible rejection reaction can be avoided in a short period, the requirements of animal experiment stages are met, and a favorable foundation is provided for developing research of central system diseases in the future.
In order to solve the technical problems, the application provides an animal model construction method of a central nervous immune cell preparation, which comprises the following steps:
s1, selecting a target animal and homologous close relatives of the target animal;
s2, performing immune cell separation, reconstruction, amplification and purification on the homologous close parents of the target animal according to the operation specification of immune cell therapy to obtain an immune cell preparation from the homologous close parents;
s3, injecting the syngeneic immune cell preparation into cerebrospinal fluid of the target animal by a intrathecal injection mode to obtain an animal model of the central nervous immune cell preparation.
Preferably, the target animal is a rodent.
Preferably, the target animal is a mouse.
Preferably, the cellular immunotherapy is selected from one or more of Polyclone Treg, CAR-Treg, TCR-Treg, CAR-T cell therapy, TCRT cell therapy and NK cell therapy.
Preferably, the intrathecal injection adopts an intrathecal injection catheter for direct administration of the immune cell therapy preparation cerebrospinal fluid, and the intrathecal injection catheter comprises a intrathecal cannula, an anchoring bead and an injection tube which are sequentially communicated, and further comprises a subcutaneous cannula (the tail end is a blind end) capable of accommodating the injection tube; the inner diameter of the intrathecal cannula gradually reduces from one end close to the anchoring bead to the other end, the maximum outer diameter of the intrathecal cannula is not more than 0.3mm, and the maximum inner diameter is not more than 0.2mm; the side of the anchoring bead near the intrathecal cannula is a rough surface.
Preferably, the anchoring bead is hemispherical or ellipsoidal, the maximum diameter of the anchoring bead not exceeding 1.5mm.
Preferably, the syringe further comprises a guide wire and a micro-syringe, and a needle opening of the micro-syringe can be inserted into the syringe.
Preferably, the minimum inner diameter of the intrathecal cannula is not less than 0.15mm.
Preferably, the inner diameter of the injection tube is 0.26-0.35 mm, and the outer diameter is 0.50-0.65 mm.
Preferably, the intrathecal cannula and the syringe are flexible tubes.
Preferably, the length of the intrathecal cannula is not less than 2cm, and the length of the injection tube is 1-3 cm.
Preferably, the anchoring beads are spherical and the roughened surface of the anchoring beads is roughened surface that can be stitched by a suture.
Compared with the prior art, the application has the beneficial effects that:
the application creatively discovers that immune cells are extracted by utilizing homologous close relatives of a target animal and processed into immune cell preparations (such as Polyclone Treg, CAR-Treg, TCR-Treg and CAR-T cells), and then the immune cell preparations are input into the target animal by means of intrathecal injection, so that rejection reaction can be not or slightly promoted in a short time, and the survival time of an animal model is prolonged, thereby meeting the requirements of animal experiments. The application innovatively and directly administrates in cerebrospinal fluid by avoiding Blood Brain Barrier (BBB), and the probability of generating rejection is further reduced due to no white blood cells or only a very small number of white blood cells in the cerebrospinal fluid. Taking the experiment of the immune cell preparation on the mouse brain tissue demyelination induced by Cuprizone as an example, the method can successfully construct a mouse model of the immune cell preparation, and carry out the following steps of biochemical analysis, pharmacological analysis and the like.
In some preferred embodiments, the present application provides for the realization of an effective animal model construction of central nervous system immune cell preparations, a special intrathecal injection catheter is designed, which has at least the following advantages:
1) Taper design: at present, the central intrathecal administration mode of mice is limited to be used for administration of small molecules, macromolecules and AAV viruses, and a slow or continuous administration mode of an osmotic pump is generally adopted. In the cell therapy, cells are required to be infused into CSF within a certain allowable time, cell viability is maintained, and a pipeline is required to be buried between two blades to avoid the accessible region of the limb of the mouse, so that the infused micro cells (such as 5 uL) are ensured to be infused into CSF as completely as possible. Therefore, the tubing must be designed with a closely fitting microinjection needle that is as straight as possible into the forward-most end of the sheath insertion tube. The sheath inner catheter adopts a taper design, and the sheath inner catheter and the injection tube are of a segment design, so that the close fit of a pipeline which is a needle head of a microinjector can be improved, and the cell preparation can be accurately guided to reach CSF through the taper design.
2) The intrathecal injection catheter is designed in a segmented way, the part (the intrathecal cannula, the anchoring bead and the injection tube) for injection and the subcutaneous tube for storage are independent, the distal end of the injection tube can be inserted into the subcutaneous tube, plugging after one-time administration is facilitated, multiple times of administration can be realized, and meanwhile, the intrathecal injection catheter is low in pollution risk, convenient to use and high in operation success rate.
3) The hemispherical or ellipsoidal anchoring beads are arranged between the intrathecal insertion tube and the injection tube, the curved rough surface of the hemispherical or ellipsoidal anchoring beads is beneficial to the horizontal entry of the intrathecal insertion tube into the sheath, and meanwhile, the stable suture fixation of the catheter at the intrathecal entrance of the spinal cord is beneficial to the maintenance of the fixed and non-rotating state of the intrathecal insertion tube, so that the damage of the microtubule movement to the spinal cord is avoided, and the stability and consistency of subsequent administration are ensured.
Drawings
FIG. 1 is a schematic view of an intrathecal injection catheter according to the present application;
FIG. 2 is a schematic view showing a combined use of the intrathecal injection catheter according to the present application;
FIG. 3 is a photograph of a mouse with an intrathecal injection catheter inserted in example 2;
wherein 1 is intrathecal cannula, 2 is injection tube, 3 is subcutaneous cannula, 4 is anchoring bead, 41 is rough surface of anchoring bead.
Detailed Description
The technical scheme of the present application will be described below with reference to the accompanying drawings. It is apparent that the described examples are only some, but not all, embodiments of the application; and the structures shown in the drawings are merely schematic and do not represent a physical object. It is intended that all other embodiments obtained by those skilled in the art based on these embodiments of the present application fall within the scope of the present application.
In the present application, the "proximal" refers to the end that is proximal to the cerebrospinal fluid of the experimental animal when in use, and the "distal" refers to the end that is distal to the cerebrospinal fluid of the experimental animal when in use. The "immune cell therapy" includes, but is not limited to, immune cell therapies such as Polyclone Treg, CAR-Treg, TCR-Treg, CAR-T class, TCRT, NK, etc., and the "immune cell therapy preparation" includes, but is not limited to, modified CAR-T cells.
The application provides a method for constructing an animal model of a central nervous immune cell preparation, which comprises the following steps:
s1, selecting a target animal and homologous close relatives of the target animal;
s2, performing immune cell separation, reconstruction, amplification and purification on the homologous close parents of the target animal according to the operation specification of immune cell therapy to obtain an immune cell preparation from the homologous close parents;
s3, injecting the syngeneic immune cell preparation into cerebrospinal fluid of the target animal by a intrathecal injection mode to obtain an animal model of the central nervous immune cell preparation.
In step S1 of the present application, the syngeneic relatives of the target animal refer to the offspring generated by mating two animals, such as mice, that are close to or from the same blood source. For example, EAE model, cuprizone-induced mouse brain tissue demyelination model, SOD1 gene mutation mouse model can be prepared, and can be used as a control by using the non-pathogenic mouse. If cells are extracted, the corresponding cells can be prepared by purification and expansion of (dried) syngeneic close-related animals without any experiment. In the present application, the target animal is preferably a rodent, more preferably a mouse.
In the step S2 of the present application, for the immune cell product to be studied, the steps of separation, amplification and characterization are performed according to the cell type characteristics, and specifically, the steps of separation, transformation, purification and the like may be performed according to various methods known in the art.
In step S3 of the present application, the present application preferably employs an intrathecal injection catheter for direct administration of an immunocytotherapy preparation into cerebrospinal fluid, as shown in fig. 1, comprising an intrathecal cannula 1, an anchor bead 4 and a syringe 2, which are sequentially connected, and a subcutaneous cannula 3 capable of receiving the syringe. The intrathecal cannula 1 is a portion inserted into the myelin sheath, the syringe 2 is used to connect a microinjector, the subcutaneous cannula 3 is used by itself buried subcutaneously to receive the exposed distal end of the syringe, and the anchor beads 4 are used to secure the device to the myelin inlet and prevent migration of the catheter. In some embodiments of the application, the intrathecal injection catheter may further comprise a guidewire and a microinjector for use in combination. In some preferred embodiments of the application, the intrathecal cannula 1, the anchoring beads 4 and the injection tube 2 are of an integrated design.
Fig. 2 shows a use mode of the intrathecal injection catheter according to the present application, after the injection is completed, the distal end of the injection tube 2 is closed and then bent, the injection tube is inserted into the subcutaneous tube 3, and then the subcutaneous tube is sealed by a sealing film or the like, and the double sealing can effectively prevent pollution, and the survival time of mice is improved, so that the multiple injections are realized.
In the present application, the intrathecal cannula 1 is designed to be tapered, the inner diameter of the intrathecal cannula 1 gradually decreases from one end close to the anchoring bead 4 to the other end, the maximum Outer Diameter (OD) of the intrathecal cannula 1 is not more than 0.3mm, and the maximum Inner Diameter (ID) is not more than 0.2mm. In some preferred embodiments of the application, the intrathecal cannula 1 is a flexible tube to accommodate bending variations within the myelin sheath. In some preferred embodiments of the present application, the length of the intrathecal cannula 1 is not less than 2cm to reach the site where injection is desired. In one embodiment of the present application, the specification of the intrathecal catheter 1 is: 2cm long, 0.254mm maximum outer diameter, 0.127mm maximum inner diameter, 0.22mm minimum outer diameter, 0.15mm minimum inner diameter, and uniform taper of inner diameter and outer diameter.
In the present application, the syringe 2 is passed through the needle of a microinjector. In some preferred embodiments of the application, the syringe 2 has a diameter of 0.26 to 0.35mm and an outer diameter of 0.50 to 0.65mm. In some preferred embodiments of the application, the syringe 2 is a flexible tube so that its distal end is bent and then placed into the subcutaneous tube. In some preferred embodiments of the application, the length of the syringe is not less than 1cm. In one embodiment of the present application, the syringe 2 is of the following specifications: 2cm long, 0.62mm outside diameter, 0.26mm inside diameter.
In the present application, the subcutaneous tube 3 is a subcutaneous tube embedded in the blind end starting from the subcutaneous end of the L3-T5. In some preferred embodiments of the application, the length of the subcutaneous tube 3 is not less than 2cm. In one embodiment of the application, the gauge of the subcutaneous tube 3 is: 2cm long, 1.22mm outside diameter, 0.27mm inside diameter.
In the present application, an anchor bead 4 is disposed between the intrathecal cannula 1 and the syringe 2, and has a diameter larger than that of the intrathecal cannula 1 and the syringe 2 to form a protrusion for fixation, generally in a hemispherical or ellipsoidal shape, and has a through hole therein for the passage of a cell reagent in the cannula. In some preferred embodiments of the application, the anchoring bead is hollow and hemispherical, the outer diameter does not exceed 1.5mm, and the inner diameter of the inner through hole is the same as that of the syringe 2. In some preferred embodiments of the present application, the rough surface 41 of the anchoring bead is a rough surface close to the hemispherical surface of the intrathecal cannula 1, and the semicircular rough surface is beneficial to the horizontal entry of the intrathecal cannula into the sheath, and simultaneously beneficial to the stable suture fixation of the catheter at the intrathecal entrance of the spinal cord, and keeps the intrathecal cannula in a fixed and non-rotating state so as to avoid damage to the spinal cord caused by movement of the microtubules and ensure the stability and consistency of subsequent administration.
In step S3 according to the present application, the intrathecal injection preferably employs the following steps:
(1) An intrathecal insertion tube of an intrathecal injection catheter is inserted from a vertebral lamina of a sixth lumbar vertebra of the experimental animal through a guide wire and is placed into a subarachnoid space;
(2) The rough surface of the anchoring bead is tightly clung to the vertebral plate downwards, and the suture line is used for suturing and fixing the peripheral muscle tissue;
(3) Placing the subcutaneous sleeve under the back of the experimental animal, and enabling the subcutaneous sleeve to be parallel to the injection tube;
(4) After the injection is completed, the distal end of the injection tube is inserted into the subcutaneous cannula after being closed, and is sealed and opened when the injection tube is used next time.
The technical solutions provided by the present application are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present application.
Example 1
For the cell products to be researched, separating and amplifying according to the characteristics of cell types; for example, for regulatory T cells, T effector cells, we will isolate, amplify, use rodent-like closely isolated cells, and administer by intrathecal injection of the environmental properties of cerebrospinal fluid.
Example 2
An intrathecal injection catheter comprises an intrathecal cannula 1 (length 2cm, max 0.254mm OD x max 0.127mm ID), an anchoring bead 4 (outer diameter 1.5 mm) and a syringe 2 (length 2cm,1.22mm OD x 0.72mm ID) which are communicated in sequence, and also comprises a subcutaneous tube 3 (length 2cm,1.22mm OD x 0.72mm ID), wherein the hemispherical surface of the anchoring bead 4 close to the intrathecal cannula 1 is rough. The intrathecal cannula 1 is of tapering design, gradually decreasing in diameter from one end proximal to the anchoring bead 4 to the inside and outside thereof.
Example 3
1. Lumbar spinal cord sheath built-in tube
1.1 laboratory glassware consumptive material
Inhalation anesthesia machine, mouse heating pad, electric dental drill, glass capillary, ophthalmic surgical instrument set, suture line, shaver, disinfectant, intrathecal injection catheter as shown in example 1, mouse brain stereotactic instrument fixing device, etc.
1.2 Experimental procedure
(1) Anesthesia
Mice were placed in an induction box and given 4% isoflurane inhalation anesthesia, and after the mice were limp, 2% isoflurane was continuously administered to maintain anesthesia after anergy. The back skin was then dehaired (prepped) with a shaver from L1-S2 and the mice were placed on a heating pad (37 ℃) to maintain body temperature. 2% iodophor is used for wiping skin at the shave part for disinfection.
(2) Exposing the spine
A longitudinal skin incision of about 1cm was made between the midlines L4-L6 of the backs of the mice with a surgical knife, the spinal processes of the sixth lumbar vertebra (L6) were identified, and the myofascial layers surrounding the L4-L6 spinal processes were blunt-separated with hemostats, exposing the L6 lamina.
(3) Tube-placing
The left hand held forceps stabilize the spine, the right hand held small electric dental drill (drill bit diameter 0.6 mm), an oval hole (0.6x1.2mm) parallel to the long axis of the spine is lightly drilled and ground on the L6 vertebral lamina, grinding is stopped when the spinal lamina is hidden to see the white spinal cord, the vertebral lamina is not completely ground through at this time, the intrathecal injection catheter (with a guide tungsten wire penetrating) shown in the embodiment 1 is used for puncturing the vertebral lamina and the dura mater, the subarachnoid space is placed, the catheter is lightly inserted into the head end, the catheter is always kept parallel to the spinal cord in the intubation process, and the catheter is slowly pushed for 2cm. Mice were seen to whip tail or tail involuntarily whip during catheterization.
After the insertion of the catheter is completed, the anchoring beads 4 are sutured with sutures to fix the muscles on both sides thereof, and the muscles are sutured layer by layer. Subcutaneous tissue of the back was blunt-separated from the skin incision (L4) of the back toward the head of the mouse with hemostats, a subcutaneous tunnel (L4-L1, length 1.0-1.5 cm) was opened, and the subcutaneous catheter 3 was implanted subcutaneously and sutured fixed. As shown in FIG. 3, the syringe 2 was inserted back into the subcutaneous tube 3 (1.0 cm) and sealed with a sealing film. Mice were intraperitoneally injected with 8 ten thousand units of penicillin to prevent infection.
When drug is again administered, the distal end of the syringe 2 is withdrawn from the subcutaneous tube 3, and a microinjector needle (0.26 mm OD) is inserted into the syringe 2 to administer the drug. After the injection is completed, the fine catheter can still be reinserted into the subcutaneous tube 3 and sealed by a sealing film.
1.3 notes
(1) The catheterization procedure must ensure that it is always parallel to the spinal cord. This facilitates insertion while reducing the risk of nerve damage.
(2) The correct catheterization process should be carried out smoothly, the pushing is not smooth in the pushing process, the phenomena of tail flicking or leg jumping of animals reflect poor insertion, and the catheter can be slightly extracted and reinserted.
2. Cerebrospinal fluid and serum collection
2.1 serum collection-retroorbital vein Cong Caixie
The procedure described is applicable to mice of any age, sex and strain weighing more than 15 g.
(1) Mice were placed in an induction box and given 4% isoflurane inhalation anesthesia, and the mice were left limp.
(2) Mice were removed from the chamber and the level of anesthesia was assessed by pedal reflex (i.e., pinching the footpad hard). Ensure sufficient depth of anesthesia before performing surgery: the lack of response to the forceful pinch indicates adequate anesthesia.
(3) The skin above the eyes was pulled back with the index finger and the skin below the eyes was pulled back with the thumb, causing the eyes to bulge.
(4) A 0.1cm inner diameter glass capillary tip was placed in the inferior inner canthus orbital at about 45 ° to the middle of the orbital. The capillary needle was slowly advanced, after resistance was encountered, the capillary was rotated, the vessel was punctured, blood was passed into the capillary, and 1.5ml of EP tubing was accessed.
Note that: the maximum amount of blood that can be drawn from this location at a time is about 1% of the body weight, for example, 0.2mL is drawn from 20g mice.
(5) The capillary was gently withdrawn, the eyelid was closed and gently pressed with gauze to prevent further bleeding. At the end of the surgery, the mice were returned to the cage.
(6) The blood was allowed to stand at Room Temperature (RT) for 30-60min and centrifuged at 2,000Xg for 10min in a centrifuge at 4 ℃. Serum was collected in a new 0.5mL EP tube using a pipette and stored frozen at-80 ℃.
2.2 cerebrospinal fluid collection-cerebellum medullary pool
(1) All used instruments and materials are sterilized prior to surgery.
(2) Mice were placed in an induction box and given 4% isoflurane inhalation anesthesia, and after the mice were limp, 2% isoflurane was continuously administered to maintain anesthesia after anergy.
(3) The prone position of the mouse is fixed on a brain stereotactic apparatus, so that the head and the body form an angle of approximately 135 degrees, and the occipital region of the mouse is sterilized and skin is prepared.
(4) A sagittal incision was made in the mid-occipital using a sterile scalpel, exposing the muscles overlying the greater foramen.
(5) Wiping with sterilized cotton ball, blunt separating subcutaneous tissue and muscle with hemostatic forceps to expose occipital macropore, and further blunt separating with cotton swab to fully expose mouse dura mater.
(6) Until the white dura mater is exposed, the surface of the dura mater is gently wiped by a sterile cotton swab, and a sample application capillary glass tube (with the inner diameter of 0.1 mm) is gently screwed under the dura mater, so that clear cerebrospinal fluid can be seen to enter the capillary.
Note that: intracranial pressure can cause spontaneous flow of cerebrospinal fluid into capillaries. About 5-12uL of CSF was obtained from each mouse depending on the age and size of the mouse.
(7) The collected CSF was injected into a 0.5mL EP tube through a capillary tube and immediately inserted into ice.
(8) The scalp is sutured using sutures.
(9) Mice were intraperitoneally injected with 8 ten thousand units of penicillin to prevent infection.
(10) CSF was centrifuged at 1,000Xg for 10min in a centrifuge at 4 ℃. The blood contaminated sample was discarded by visual inspection for blood contamination.
(11) PBS was used at 1:3 diluting cerebrospinal fluid to reduce the volume loss caused by aerosols. Immediately, CSF vials were frozen at-80 ℃.
2.3 collection of serum and cerebrospinal fluid at the end of the test
Note that: for the end-stage live fluid collection of the test, cerebrospinal fluid collection needs to precede serum collection because mice need to have pulses before cerebrospinal fluid can be collected.
2.3.1 cerebrospinal fluid Collection at necropsy
Note that: this procedure was suitable for non-viable surgery, obtaining about 10-20uL CSF from each mouse. Sterile surgical fields are recommended, but are not required for non-viable surgery.
2.3.2 blood collection by intracardiac puncture (open method)
Note that: the expected blood volume is approximately 3% of body weight, e.g., 0.6mL for 20g mice.
After cerebrospinal fluid collection, the mice were ensured to remain fully anesthetized by pinching the footpad. If any response is observed, a second anesthetic is administered. If no reaction is observed, please continue.
The animals were placed on the back and the abdominal skin was rubbed with 70% alcohol. The chest was opened with surgical scissors to expose the heart. A 25G needle (connected to a 3mL syringe) was inserted into the left ventricle and a gentle negative pressure was applied to the syringe plunger. After blood collection, the needle is pulled out.
Secondary methods of euthanasia, such as chop or cervical dislocation, are performed to ensure death of the animal.
The plunger of the syringe was pushed down and the collected blood was injected into a 1.5mL vial. The blood was allowed to coagulate at room temperature for 30-60 minutes and then centrifuged at 2,000Xg for 10 minutes in a refrigerated centrifuge at 4 ℃.
Serum was collected into a new labeled 0.5mL vial using a clean pipette. The serum bottles were immediately frozen in a-80 ℃ refrigerator.
The foregoing is merely a preferred embodiment of the present application and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present application, which are intended to be comprehended within the scope of the present application.
Claims (12)
1. The animal model construction method of the central nervous immune cell preparation is characterized by comprising the following steps of:
s1, selecting a target animal and homologous close relatives of the target animal;
s2, performing immune cell separation, reconstruction, amplification and purification on the homologous close parents of the target animal according to the operation specification of immune cell therapy to obtain an immune cell preparation from the homologous close parents;
s3, injecting the syngeneic immune cell preparation into cerebrospinal fluid of the target animal by a intrathecal injection mode to obtain an animal model of the central nervous immune cell preparation.
2. The method of claim 1, wherein the target animal is a rodent.
3. The method of claim 2, wherein the target animal is a mouse.
4. The animal model construction method according to claim 1, wherein the cellular immunotherapy is selected from one or more of Polyclone Treg, CAR-Treg, TCR-Treg, CAR-T cell therapy, TCRT cell therapy and NK cell therapy.
5. The method of claim 1, wherein the intrathecal injection is performed using an intrathecal injection catheter for direct administration of the immune cell therapy formulation into the cerebrospinal fluid,
the intrathecal injection catheter comprises a intrathecal cannula, an anchoring bead and a syringe which are sequentially communicated, and further comprises a subcutaneous cannula capable of accommodating the syringe;
the inner diameter of the intrathecal cannula gradually reduces from one end close to the anchoring bead to the other end, the maximum outer diameter of the intrathecal cannula is not more than 0.3mm, and the maximum inner diameter is not more than 0.2mm;
the side of the anchoring bead near the intrathecal cannula is a rough surface.
6. The method according to claim 5, wherein the anchor beads are hemispherical or ellipsoidal, and the maximum diameter of the anchor beads is not more than 1.5mm.
7. The method of claim 5, further comprising a guide wire and a microinjector, a needle port of which is insertable into the syringe.
8. The method according to claim 5, wherein the minimum inner diameter of the intrathecal cannula is not less than 0.15mm.
9. The method according to claim 5, wherein the syringe has an inner diameter of 0.26 to 0.35mm and an outer diameter of 0.50 to 0.65mm.
10. The method according to claim 5, wherein the intrathecal cannula and the syringe are flexible tubes.
11. The method according to claim 5, wherein the length of the intrathecal cannula is not less than 2cm and the length of the injection tube is 1 to 3cm.
12. The method according to claim 5, wherein the anchor beads are spherical, and the roughened surface of the anchor beads is a roughened surface that can be sewn by a suture.
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