Culture medium optimization method based on Raman spectrum and cell culture medium

Abstract

The invention belongs to the technical field of cell culture, and particularly relates to a Raman spectrum-based culture medium optimization method and a cell culture medium, the method comprises the following steps: (1) adding different amounts of effective components into a basic culture medium to prepare a plurality of cell culture mediums, the basic culture medium does not contain the effective components; (2) respectively culturing the cells by adopting the plurality of cell culture mediums to obtain different culture solutions containing the cells; and (3) respectively placing the different culture solutions containing the cells under a Raman microscope, and collecting Raman spectrums of the cells in the different culture solutions so as to obtain the optimized cell culture medium. According to the culture medium optimization method provided by the invention, the Raman spectrum is adopted for detecting cells in the culture medium for the first time so as to optimize the dosage of effective components in the cells, and the optimized culture medium can meet the requirements of the effective components for cell growth and cannot cause waste of the effective components.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for optimizing culture medium based on Raman spectroscopy and a cell culture medium. Background Technology

[0002] The fall armyworm (Spodoptera frugiperda) is a species of moth belonging to the genus Spodoptera in the family Noctuidae of the order Lepidoptera. Its cells are a commonly used insect cell in biological experiments, often referred to as Sf9 cells. These cells are typically used as an insect platform to produce various proteins and viruses, or to conduct basic research.

[0003] In Sf9 cell culture, the composition of the culture medium plays a significant role. Cholesterol, as an important upstream substance for hormone synthesis, greatly affects cell number and state. Traditionally, based on previous research, the amount of cholesterol added is usually controlled at a final concentration of around 2-5 μg / mL. Currently, there are few reported methods on how to optimize the amount of cholesterol added to the culture medium. Summary of the Invention

[0004] In view of this, the present invention provides a method for optimizing culture media based on Raman spectroscopy, thereby solving or at least alleviating one or more of the above-mentioned problems and other problems existing in the prior art.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for optimizing culture media based on Raman spectroscopy includes the following steps: (1) Adding different amounts of the active ingredient to the basal culture medium to prepare multiple cell culture media, wherein the basal culture medium does not contain the active ingredient; (2) The cells are cultured separately using the multiple cell culture media to obtain different culture media containing cells; (3) Place the different culture media containing cells under a Raman microscope and collect the Raman spectra of the cells in the different culture media to obtain an optimized cell culture medium.

[0006] In the aforementioned Raman spectroscopy-based culture medium optimization method, the active ingredient may optionally be cholesterol.

[0007] The cholesterol mentioned is also known as cholesterolsterol, with the chemical formula C60. 27 H 46 O, CAS number 57-88-5.

[0008] Optionally, the method further includes step (3) observing the morphology of cells in different culture media using a Raman microscope.

[0009] Optionally, the Raman spectra of cells in the optimized cell culture medium are in the range of 2800~3000 cm⁻¹. -1 It has a characteristic peak with an intensity of over 6000, and the cell membrane in the optimized cell culture medium is intact and has a smooth surface.

[0010] In the aforementioned Raman spectroscopy-based culture medium optimization method, optionally, the cholesterol concentration in the optimized cell culture medium is 0.3~0.65 μg / mL. For example, the cholesterol concentration in the optimized cell culture medium may be 0.3 μg / mL, 0.35 μg / mL, 0.4 μg / mL, 0.45 μg / mL, 0.5 μg / mL, 0.55 μg / mL, 0.6 μg / mL, 0.65 μg / mL, etc.

[0011] Further optionally, the cholesterol concentration in the optimized cell culture medium is 0.4~0.65 μg / mL.

[0012] In the aforementioned Raman spectroscopy-based culture medium optimization method, optionally, the cells are fall armyworm cells; and the basal culture medium is Sf9 insect cell culture medium.

[0013] Optionally, the basal culture medium is InsectPro. ® Sf9 insect cell serum-free culture medium II.

[0014] In the aforementioned Raman spectroscopy-based culture medium optimization method, optionally, in step (1), the preparation method of multiple cell culture media includes: adding different proportions of effective components to the basic culture medium to prepare a cell culture medium with a final concentration of effective components of 0-50 μg / mL.

[0015] In the aforementioned Raman spectroscopy-based culture medium optimization method, optionally, the culture conditions in step (2) are 27±2℃ and the culture time is 24±2h.

[0016] The second technical solution adopted in this invention is: a cell culture medium, wherein the cells are Sf9 cells, the cell culture medium includes a basal culture medium and cholesterol, wherein the basal culture medium does not contain cholesterol, and the final concentration of cholesterol is 0.4~0.65μg / mL.

[0017] Optionally, the basal culture medium is Sf9 insect cell culture medium.

[0018] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention provides a method for optimizing culture media. For the first time, Raman spectroscopy is used to detect cells in the culture media to optimize the amount of effective components. The optimized culture media can meet the needs of effective components for cell growth without wasting effective components.

[0019] The optimization method of this invention is economical, real-time, and fast. Attached Figure Description

[0020] Figure 1 The image shows the Raman spectra of Sf9 cells cultured in a cell culture medium with a cholesterol concentration of 0.6 μg / mL.

[0021] Figure 2 Microscopic images of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0.6 μg / mL.

[0022] Figure 3 The image shows the Raman spectrum of Sf9 cells cultured in a cell culture medium with a cholesterol concentration of 0.3 μg / mL.

[0023] Figure 4 Microscopic images of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0.3 μg / mL.

[0024] Figure 5 The image shows the Raman spectrum of Sf9 cells after being cultured in a cell culture medium with a cholesterol concentration of 0 μg / mL.

[0025] Figure 6 Microscopic images of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0 μg / mL. Detailed Implementation

[0026] Raman spectroscopy is a type of scattering spectroscopy. Based on the Raman scattering effect discovered by Indian scientist CV Raman, Raman spectroscopy analyzes the scattered spectra with frequencies different from the incident light to obtain information about molecular vibrations and rotations, and is applied to molecular structure research.

[0027] In Sf9 cell culture, Sf9 cells exhibit different characteristics in environments with different cholesterol concentrations. After cell detection using Raman spectroscopy, the proportion of cholesterol added can be determined by determining the cell state, thus completing the determination and optimization of the main components of the culture medium, and obtaining an optimized cell culture medium through this optimization method.

[0028] This invention provides a method for optimizing culture media based on Raman spectroscopy, comprising the following steps: (1) Add different amounts of active ingredients to the basal culture medium to prepare multiple cell culture media, wherein the basal culture medium does not contain active ingredients; (2) Cells were cultured separately using multiple cell culture media to obtain different culture media containing cells; (3) Place different culture media containing cells under a Raman microscope and collect the Raman spectra of cells in different culture media to obtain an optimized cell culture medium.

[0029] In some alternative embodiments, the active ingredient is cholesterol; in other embodiments, the active ingredient may be other major components of the culture medium.

[0030] In some alternative embodiments, the method further includes step (3) observing the morphology of cells in different culture media using a Raman microscope. If the cell membrane is intact and the surface is smooth, there are no bright spots or the bright spots are negligible in the image (e.g., Figure 2 If the cell surface is uneven, bright spots will appear in the image (e.g., Figure 4 and Figure 6 ).

[0031] In some alternative embodiments, in step (3), the Raman spectra of cells in the optimized cell culture medium are at Raman shifts of 2800~3000 cm⁻¹. -1 It has characteristic peaks with an intensity of over 6000, and the cell membranes in the optimized cell culture medium are intact and have smooth surfaces.

[0032] The effective Raman shift peak range in the Raman spectrum is 500-3000 cm⁻¹. -1 .

[0033] In some alternative embodiments, the cholesterol concentration in the optimized cell culture medium is 0.3-0.65 μg / mL, further 0.4-0.65 μg / mL, and even further 0.5-0.65 μg / mL.

[0034] In some alternative embodiments, the cells are fall armyworm cells; the basal culture medium is Sf9 insect cell culture medium, such as InsectPro. ® Sf9 insect cell serum-free culture medium II, animal-free.

[0035] In some optional embodiments, in step (1), the method for preparing multiple cell culture media includes: adding different proportions of effective ingredients to the basic culture medium to prepare a cell culture medium with a final concentration of effective ingredients of 0-50 μg / mL, such as 0 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.25 μg / mL, 2.5 μg / mL, etc.

[0036] In some alternative embodiments, the culture conditions for step (2) are 27±2℃ and the culture time is 24±2h.

[0037] A second aspect of the present invention also provides an optimized cell culture medium, wherein the cells are Sf9 cells, and the cell culture medium includes a basal medium and cholesterol, wherein the basal medium is cholesterol-free and the final cholesterol concentration is 0.4~0.65 μg / mL.

[0038] In some alternative embodiments, the final cholesterol concentration is 0.5-0.65 μg / mL.

[0039] The present invention will be further described below through specific embodiments.

[0040] Example 1 I. Preparation of Cell Culture Media 1. Raw materials Active ingredient: Cholesterol stock solution (4mg / mL); Basic culture medium: InsectPro ® Sf9 insect cell serum-free culture medium II, animal-free (cholesterol-free), hereinafter referred to as InsectPro ® Sf9 culture medium; 2. Configuration Method Take the cholesterol stock solution and, based on the required basal culture medium volume and cholesterol concentration, transfer a predetermined amount of cholesterol stock solution to 1000 mL of InsectPro. ® Sf9 medium was diluted to target cholesterol concentrations (0 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.25 μg / mL, 2.5 μg / mL), mixed thoroughly, and dispensed into 5 sample cell culture media.

[0041] II. Cell Culture 1. Use cholesterol-free and serum-free basal culture medium for subculturing, and ensure aseptic conditions throughout the entire culture process.

[0042] Take Sf9 cells into a 50mL centrifuge tube, and add basal culture medium to a final volume of 45mL, so that the cell density is (0.5~1.0)×10⁻⁶. 6 cells / mL (e.g., 0.6 × 10⁻⁶) 6 Then mix using a Pasteur pipette (cells / mL).

[0043] Add 10 mL of InsectPro to a 9 mm diameter culture dish. ® Sf9 medium and 1 mL of basal medium containing Sf9 cells were incubated overnight at 27°C.

[0044] 2. Cell culture was performed using the culture media from the above five sample cells. Using a pipette, 10 mL of culture medium containing Sf9 cells was drawn and added to five sample cell culture media containing 0 μg / mL, 0.3 μg / mL, 0.6 μg / mL, 1.25 μg / mL, and 2.5 μg / mL cholesterol, respectively. The cells were then incubated at 27°C for 24 h.

[0045] result: Five cultured cell samples (100 μL each) were added to 96-well plates, followed by 100 μL of sterile phosphate buffer. The plates were then placed under a Raman microscope at room temperature to observe cell morphology. Raman spectroscopy data of Sf9 cells were recorded. The results are as follows: Figures 1-6 As shown.

[0046] in, Figure 1 The image shows the Raman spectra of Sf9 cells cultured in a cell culture medium with a cholesterol concentration of 0.6 μg / mL. Figure 2 Microscopic images of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0.6 μg / mL.

[0047] Figure 3 The image shows the Raman spectra of Sf9 cells cultured in a cell culture medium with a cholesterol concentration of 0.3 μg / mL. Figure 4 Microscopic images of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0.3 μg / mL.

[0048] Figure 5 The image shows the Raman spectra of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0 μg / mL. Figure 6 Microscopic images of Sf9 cells cultured in cell culture medium with a cholesterol concentration of 0 μg / mL.

[0049] As shown above, in cell culture media with a cholesterol concentration of 0.3 μg / mL or higher, the characteristic peak intensity of cholesterol in the Raman spectrum of Sf9 cells reached over 6000. However, microscopic images revealed that the cell membranes in the 0.3 μg / mL medium were not sufficiently intact, and the surface was not smooth enough, resulting in numerous bright spots in the images. In contrast, the cell membranes in the 0.6 μg / mL medium were intact, the surface was smooth, and no bright spots were observed in the images. This indicates that 0.3 μg / mL of cholesterol is insufficient, while 0.6 μg / mL is sufficient for cell growth.

[0050] This further indicates that cholesterol concentrations do not need to be too high, such as 1.2 μg / mL or even higher.

[0051] The above embodiments are only used to illustrate the embodiments of the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the embodiments of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the embodiments of the present invention, and the patent protection scope of the embodiments of the present invention should be defined by the claims.

Claims

1. A method for optimizing culture media based on Raman spectroscopy, characterized in that, The method includes the following steps: (1) Adding different amounts of the active ingredient to the basal culture medium to prepare multiple cell culture media, wherein the basal culture medium does not contain the active ingredient; (2) The cells are cultured separately using the multiple cell culture media to obtain different culture media containing cells; (3) Place the different culture media containing cells under a Raman microscope and collect the Raman spectra of the cells in the different culture media to obtain an optimized cell culture medium.

2. The method for optimizing culture medium based on Raman spectroscopy according to claim 1, characterized in that, The active ingredient is cholesterol.

3. The method for optimizing culture medium based on Raman spectroscopy according to claim 2, characterized in that, The method also includes step (3), in which the morphology of cells in different culture media is observed by Raman microscopy.

4. The method for optimizing culture medium based on Raman spectroscopy according to claim 3, characterized in that, The Raman spectra of cells in the optimized cell culture medium were in the Raman shift range of 2800–3000 cm⁻¹. -1 It has a characteristic peak with an intensity of over 6000, and the cell membrane in the optimized cell culture medium is intact and has a smooth surface.

5. The method for optimizing culture medium based on Raman spectroscopy according to claim 2, characterized in that, The cholesterol concentration in the optimized cell culture medium is 0.3~0.65 μg / mL.

6. The method for optimizing culture medium based on Raman spectroscopy according to claim 5, characterized in that, The cholesterol concentration in the optimized cell culture medium is 0.4~0.65 μg / mL.

7. The method for optimizing culture media based on Raman spectroscopy according to claim 1, characterized in that, The cells were fall armyworm cells; the basal culture medium was Sf9 insect cell culture medium.

8. The method for optimizing culture medium based on Raman spectroscopy according to claim 7, characterized in that, The basal culture medium is InsectPro. ® Sf9 insect cell serum-free culture medium II.

9. The method for optimizing culture media based on Raman spectroscopy according to claim 1, characterized in that, In step (1), the preparation methods for multiple cell culture media include: adding different proportions of active ingredients to the basal culture medium to prepare cell culture media with a final concentration of active ingredients of 0-50 μg / mL; and / or, The culture conditions for step (2) are 27±2℃ and the culture time is 24±2h.

10. A cell culture medium, wherein the cells are Sf9 cells, characterized in that, The cell culture medium includes a basal culture medium and cholesterol, wherein the basal culture medium does not contain cholesterol, and the final concentration of cholesterol is 0.4~0.65 μg / mL.

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