A method for long-term in vitro expansion and culture of primary mouse alveolar macrophages

Abstract

The present invention belongs to the field of cell culture technology, and particularly relates to a method for culturing primary mouse alveolar macrophages for long-term in vitro expansion. The method uses Macrophage-SFM serum-free culture medium containing 20-40 ng / ml GM-CSF, 5-15 ng / ml TGFβ and 0.8-1.2 μM rosiglitazone to culture the isolated primary mouse alveolar macrophages, thereby achieving long-term expansion and culture of primary mouse alveolar macrophages.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a culture method for long-term in vitro expansion of primary mouse alveolar macrophages. Background Art

[0002] Macrophages play a key role in antimicrobial defense, tissue damage repair, and anti-tumor. In vitro, the cytokine M-CSF is used to induce bone marrow precursor cells or blood monocytes to differentiate into macrophages, which are called bone marrow-derived macrophages or monocyte-derived macrophages. These in vitro induced macrophages are often used to study the functions of primary macrophages. However, these macrophages have a limited lifespan (7-10 days) and need to be constantly re-induced to meet the needs of long-term research.

[0003] The existing research method is to culture alveolar macrophages in vitro with complete culture medium containing 10% fetal bovine serum and GM-CSF. Because fetal bovine serum contains exogenous substances of uncertain composition, not all serum-containing culture medium can achieve long-term culture of alveolar macrophages, so this method requires testing different batches of fetal bovine serum to find serum suitable for long-term culture. Serum screening is time-consuming and labor-intensive, and requires a lot of financial support.

[0004] For example, in the prior art, Yun Hongfang et al. (Experimental study on in vitro culture of mouse alveolar macrophages under different conditions, "Chinese Journal of Immunology", Vol. 40, 2024, pp. 1121-1125) disclosed an in vitro culture method for alveolar macrophages (AMs), using GM-CSF, TGF-β and PPAR-γ agonist rosiglitazone (GTR for short) culture system to culture and analyze the characteristics of AM-like cells or AMs produced by precursor cells of different ages and different sources. It uses serum-containing culture medium such as DMEM complete culture medium, and the culture time is 9 days, which is a short-term culture.

[0005] Another example is the prior art CN109082411B, which discloses a method for inducing the differentiation of induced pluripotent stem cells or embryoid bodies into macrophages. The first six stages all use serum-free culture medium, but the last stage uses serum culture medium for the proliferation of macrophages. The culture time is also only 45 days throughout the whole process, and serum culture medium is used from the 29th day of culture to facilitate the proliferation of macrophages.

[0006] Therefore, it is necessary to find a more reliable and stable culture system that can culture and expand alveolar macrophages for a long time. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to propose a method for culturing primary mouse alveolar macrophages for long-term in vitro expansion. By adding GM-CSF, TGFβ and rosiglitazone to the serum-free culture medium, the problem of short in vitro culture time of alveolar macrophages and the need to screen specific serum-containing culture medium for long-term in vitro culture and expansion is solved.

[0008] To achieve the above objectives, on the one hand, the present invention provides a method for long-term in vitro expansion and culture of mouse primary alveolar macrophages, wherein the isolated mouse primary alveolar macrophages are expanded and cultured in Macrophage-SFM serum-free medium containing 20-40 ng / ml GM-CSF, 5-15 ng / ml TGFβ and 0.8-1.2 μM rosiglitazone.

[0009] Furthermore, in the in vitro long-term expansion and culture method of mouse primary alveolar macrophages, the concentrations of GM-CSF, TGFβ and rosiglitazone are 30 ng / ml, 10 ng / ml and 1 μM, respectively.

[0010] Furthermore, in the in vitro long-term expansion and culture method of mouse primary alveolar macrophages, the mouse primary alveolar macrophages are alveolar macrophages isolated and obtained after mouse alveolar lavage.

[0011] Furthermore, the in vitro long-term expansion and culture method of mouse primary alveolar macrophages, wherein the long-term expansion and culture refers to culture for more than 45 days, 48 ​​days, 60 days, 70 days, 80 days or 90 days.

[0012] Furthermore, the in vitro long-term expansion and culture method of mouse primary alveolar macrophages, wherein the long-term expansion and culture means that the macrophages can still maintain proliferation.

[0013] The advantage of our method is that the components of the serum-free culture medium used are stable and there is no batch effect, so there is no need to spend time and money to find serum suitable for in vitro culture of primary alveolar macrophages. In addition, the serum-free culture medium does not contain foreign proteins. When studying the in vivo therapeutic effect of primary alveolar macrophages, it is safer and lays the foundation for future clinical research on the use of primary alveolar macrophages to treat lung diseases.

[0014] We have established a method for long-term culture and large-scale expansion of primary alveolar macrophages in vitro. Here, we used Macrophage-SFM serum-free medium and GM-CSF / TGFβ / Rosiglitazone to culture freshly isolated mouse alveolar macrophages in vitro. We found that this culture system can expand alveolar macrophages in vitro for a long time, and after transplantation into alveolar macrophage-deficient mice, these in vitro cultured alveolar macrophages can survive for a long time in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Freshly isolated alveolar macrophages were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) and GM-CSF (30 ng / ml). The number (A) and morphological changes (B) of alveolar macrophages at different time points after culture.

[0016] Figure 2 Screening and comparison of serum-free culture media. SFM represents the use of Macrophage-SFM (Gibco) serum-free culture medium to culture alveolar macrophages; X-VIVO represents the use of X-VIVO 15 (Lonza) serum-free culture medium to culture alveolar macrophages.

[0017] Figure 3 . The morphology and number of alveolar macrophages under different culture conditions. (A: Cell photos 40× on the 6th and 11th days of SFM culture. B: Changes in the number of alveolar macrophages under different culture conditions).

[0018] Figure 4 . Flow cytometry was used to detect the expression of marker molecules on the surface of alveolar macrophages under different stimulation conditions.

[0019] Figure 5 In vitro proliferation of alveolar macrophages from different donor mice in the presence of GM-CSF (30 ng / ml), TGFβ (10 ng / ml) and rosiglitazone (1 μM).

[0020] Figure 6 . The morphology of primary alveolar macrophages cultured in Macrophage-SFM (Gibco) medium with GM-CSF (30 ng / ml), TGFβ (10 ng / ml) and rosiglitazone (1 μM) for 93 days (D93). Magnifications are 20× and 40×, respectively.

[0021] Figure 7. GFP+ exAMs cultured in vitro were transplanted into the lungs of Rxrafl / flCsf1rCre mice by intratracheal injection. Donor GFP+ exAMs were detected 45 days later. DETAILED DESCRIPTION

[0022] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0023] Example 1: Obtaining alveolar macrophages by primary alveolar lavage of mice

[0024] In order to establish a suitable in vitro culture system for primary alveolar macrophages (AMs), we first lavaged the alveoli of mice and cultured the obtained alveolar macrophages in vitro.

[0025] 1. Prepare a 15 ml centrifuge tube containing 3 ml of complete culture medium for each C57 mouse.

[0026] 2. Warm the BALF buffer to 37°C in a water bath.

[0027] 3. Intraperitoneal injection of anesthetic (anesthetic: Afotin, produced by Sigma).

[0028] 4. Use dissecting tools to remove the skin, rib cage, and muscle to expose the lungs and trachea, avoiding cutting or damaging blood vessels.

[0029] 5. Using fine scissors, make a small incision in the upper part of the trachea just below the larynx.

[0030] 6. Use the incision to insert a slightly blunted 18G cannula and guide the cannula 5 mm deep into the trachea.

[0031] 7. Connect a 1 mL syringe filled with 1 mL of warm BALF buffer to the inserted cannula.

[0032] 8. Inject 1 ml of buffer while holding the cannula in place with your other hand.

[0033] 9. Pull the plunger to collect the BALF into the syringe. Approximately 800-900 μl should be recovered. After injection and collection, the lungs should visibly expand and deflate.

[0034] 10. Filter the collected BALF through a 70 μm cell strainer into the 15 ml tube prepared in step 1.

[0035] 11. Repeat the alveolar lavage twice and collect the alveoli into the same 15 ml centrifuge tube.

[0036] 12. Collect the cells by centrifugation at 500 g for 5 minutes at 4°C. Discard the supernatant.

[0037] 13. Resuspend the cell pellet in 500 μl of bronchoalveolar lavage buffer. Stain with trypan blue and count the cells. Count only live cells (trypan blue negative).

[0038] 14. Count the total number of cells in each bronchoalveolar lavage fluid.

[0039] 15. 3-5×10 per well 5 Alveolar macrophages were plated in untreated 6-well plates with 2.5 ml of culture medium per well.

[0040] 16. When plating freshly isolated primary alveolar macrophages, add gentamicin (1:1000) to the culture medium.

[0041] 17. After 6-18 hours, replace the medium with freshly prepared culture medium.

[0042] 18. After that, the cell culture medium was changed every 2-3 days. Primary alveolar macrophages derived from mouse bronchoalveolar lavage were obtained.

[0043] Bronchoalveolar lavage fluid (BAL buffer) formula: 1×PBS, 2mM EDTA (Sinopharm), 0.5% fetal bovine serum (FBS, Gibco)

[0044] Culture medium, cytokines and agonists: Macrophage-SFM (Gibco), 100x penicillin / streptomycin (Thermo), fetal bovine serum (FBS, Gibco), RPMI-1640 (EallBio), GM-CSF (30 ng / ml, NearBio), TGFβ (10 ng / ml, NearBio), rosiglitazone (1 μM, Selleck).

[0045] Example 2: RPMI-1640 complete medium containing fetal bovine serum cannot expand alveolar macrophages in vitro for a long time

[0046] Primary alveolar macrophages obtained from mouse bronchoalveolar lavage were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum (FBS) containing GM-CSF (30ng / ml). We found that the number of alveolar macrophages doubled during 4-19 days of in vitro culture; however, the cell proliferation rate slowed down on the 41st day of in vitro culture. Trypan blue staining showed that the cell viability was low (45%-70%); on the 49th day of culture, we found that the number of cells decreased significantly, and the proportion of cells with a high degree of differentiation was high ( Figure 1 A and 1B). There have been literatures suggesting that long-term in vitro culture of alveolar macrophages requires screening of different batches of fetal bovine serum. From our results, it may be that the fetal bovine serum used in this experiment is not suitable for long-term in vitro culture of alveolar macrophages.

[0047] Example 3: Validation and screening of primary alveolar macrophages cultured in serum-free medium

[0048] To screen serum-free culture media, we selected two serum-free culture media, X-VIVO 15 (Lonza) and Macrophage-SFM (Gibco) (supplemented with 100× penicillin / streptomycin (Thermo)), to which GM-CSF, GM-CSF / TGFβ, GM-CSF / rosiglitazone, and GM-CSF / TGFβ / rosiglitazone were added, respectively.

[0049] The applied concentrations of GM-CSF, TGFβ, and rosiglitazone were 30ng / ml, 10ng / ml, and 1μM, respectively. Afterwards, we used the above-mentioned culture medium containing different additives to culture freshly isolated primary mouse alveolar macrophages. The serum-free culture medium containing the above-mentioned additives was replaced every 2-3 days. X-VIVO 15 (Lonza) and Macrophage-SFM (Gibco) serum-free culture media were selected, and different combinations of GM-CSF, TGFβ, and rosiglitazone were set up to culture alveolar macrophages. We found that on the 6th day of in vitro culture, a large number of cells in the X-VIVO 15 culture group died, while the cells in the Macrophage-SFM culture group were in good condition ( Figure 2 On day 11, the cell density and number of the Macrophage-SFM culture group (Triple) containing GM-CSF, TGFβ, and rosiglitazone were the highest ( Figure 3 A and 3B). At the same time, we used flow cytometry to detect the expression of marker molecules on the surface of alveolar macrophages and found that all three stimulation combinations could maintain the expression of CD11c and SiglecF by alveolar macrophages ( Figure 4These results suggest that the application of Macrophage-SFM serum-free medium containing GM-CSF, TGFβ and rosiglitazone can maintain the long-term survival and expansion of primary alveolar macrophages.

[0050] Example 4: Long-term culture of alveolar macrophages in serum-free medium

[0051] We isolated alveolar macrophages from 5 mice (same method as in Example 1). Prepare Macrophage-SFM (Gibco) serum-free medium containing GM-CSF / TGFβ / rosiglitazone (supplemented with 100× penicillin / streptomycin (Thermo)). The concentrations of GM-CSF, TGFβ and rosiglitazone used were 30 ng / ml, 10 ng / ml and 1 μM, respectively. We used this medium containing the above additives to culture freshly isolated primary mouse alveolar macrophages. The serum-free medium containing the above additives was replaced every 2-3 days.

[0052] It was found that these five primary alveolar macrophages from different individuals were able to survive for a long time and proliferate about 40 times ( Figure 5 ), which has been cultured to 93 days ( Figure 6 ), and can still be cultured. This indicates that the method of long-term culture of alveolar macrophages in vitro has been successfully established.

[0053] Example 5: In vivo transplantation of alveolar macrophages expanded in vitro

[0054] We anesthetized the mice by intraperitoneal injection of anesthetics, placed the mice with their backs facing the operator, and used an incisor ring to suspend the upper incisors and fix them on a hanging table. Forceps were used to pull the tongue of the mice to expose their throats. The chest cavity of the mice was illuminated by a light and the position was adjusted until the glottis of the mice was observed (this step can also be achieved using a small animal laryngoscope). A retention needle (24GA) was inserted into the trachea through the glottis. After the needle was removed, an appropriate volume (2×10 6 Cells in 50-80μlPBS) cell suspension, GFP-positive ex vivo expanded alveolar macrophages (exAMs) were transplanted into alveolar macrophage-deficient mice ( RxR fl / fl Csf1 Cre ) of the lungs.

[0055] Intratracheal injection method: After intraperitoneal injection of anesthetic to anesthetize the mouse, the mouse is placed with its back facing the operator, and the upper incisors are suspended on a hanging table with an incisor ring. The tongue of the mouse is pulled with forceps to expose its throat. The mouse chest is illuminated with a light and the position is adjusted until the mouse glottis is observed (this step can also be achieved using a small animal laryngoscope). A retention needle (24GA) is inserted into the trachea through the glottis. After the needle is removed, an appropriate volume (2×10 6 The GFP-positive ex vivo expanded aleveolar macrophages (exAMs) were transplanted into the lungs of alveolar macrophage-deficient mice (Rxrafl / flCsf1rCre).

[0056] 45 days after transplantation, we prepared mouse lung cell suspensions, used flow cytometry antibodies (CD45, SiglecF, CD11c) to label mouse alveolar macrophages, and used flow cytometry to detect the proportion of GFP-positive cells in CD45+SiglecF+CD11c+ alveolar macrophages to determine whether exAMs survived after transplantation. We found that exAMs can survive for a long time in the lungs of alveolar macrophage-deficient mice ( Figure 7 ), which suggests the possibility of using alveolar macrophages cultured in vitro for in vivo cell therapy.

[0057] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A method for long-term in vitro expansion and culture of primary mouse alveolar macrophages, characterized in that: The isolated mouse primary alveolar macrophages were expanded and cultured in Macrophage-SFM serum-free medium containing 20-40 ng / ml GM-CSF, 5-15 ng / ml TGFβ and 0.8-1.2 μM rosiglitazone.

2. The in vitro long-term expansion and culture method of mouse primary alveolar macrophages according to claim 1, characterized in that: The concentrations of GM-CSF, TGFβ and rosiglitazone were 30 ng / ml, 10 ng / ml and 1 μM, respectively.

3. The in vitro long-term expansion and culture method of mouse primary alveolar macrophages according to claim 1, characterized in that: The mouse primary alveolar macrophages are alveolar macrophages separated and obtained after mouse alveolar lavage.

4. The in vitro long-term expansion and culture method of mouse primary alveolar macrophages according to claim 1, characterized in that: The long-term expansion culture refers to culture for more than 45 days.

5. The in vitro long-term expansion and culture method of mouse primary alveolar macrophages according to claim 2 or 3, characterized in that: The long-term expansion culture refers to culture for more than 48 days, 60 days, 70 days, 80 days or 90 days.

6. The in vitro long-term expansion and culture method of mouse primary alveolar macrophages according to claim 5, characterized in that: The long-term expansion culture means that the macrophages can still maintain proliferation.

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