Assembly for cell culture and bio-optical detection

Abstract

The present application relates to an assembly for cell culture and biologic optical detection, the assembly comprising a cell culture vessel having a light transparent bottom and a substrate removably attached to the light transparent bottom of the cell culture vessel, at least a portion of the surface of the substrate opposite the light transparent bottom of the cell culture vessel being light non-transparent.

Description

Technical Field

[0001] This application relates to a component for cell culture and bio-optical detection. Background Technology

[0002] Cell biology research frequently employs various bio-optical detection methods based on chemiluminescence or fluorescence. For example, luciferase is introduced into cells as a reporter molecule for transgenesis, and the expression level of the transgene is evaluated based on the intensity of biofluorescence induced by luciferase. Furthermore, before, during, or after bio-optical testing, microscopic observation of cell quantity or biological morphology is often required, necessitating cell culture containers with light transmittance suitable for microscopic observation.

[0003] The translucency of cell culture container materials may lead to light loss in bioluminescence, and whether this affects the performance of bio-optical assays is not reported in the art. If such an effect exists, improved experimental tools for cell culture and bio-optical assays are needed in the field. Summary of the Invention

[0004] In a first aspect, this application provides a component for cell culture and bio-optical detection, the component comprising: A cell culture container having a light-transmitting bottom (e.g., a colorless, light-transmitting bottom) and optionally, having opaque sidewalls (e.g., white, opaque sidewalls). A substrate, detachably attached to the light-transmitting bottom of the cell culture vessel, wherein at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is opaque, and the color of this portion of the surface is defined according to the CMYK standard as follows: C: 0.0389 to 0.0414; M: 0.0794 to 0.1485; Y: 0.3114 to 0.7303; K: 0.0576 to 0.0849.

[0005] In some embodiments of the first aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the CMYK standard as: C: 0.0389; M: 0.0794; Y: 0.3114; K: 0.0576.

[0006] In some embodiments of the first aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the CMYK standard as: C: 0.0414; M: 0.1485; Y: 0.7303; K: 0.0849.

[0007] Secondly, this application provides a component for cell culture and bio-optical detection, the component comprising: A cell culture container having a light-transmitting bottom (e.g., a colorless, light-transmitting bottom) and optionally, having opaque sidewalls (e.g., white, opaque sidewalls). A substrate, detachably attached to the light-transmitting bottom of the cell culture vessel, wherein at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is opaque, and the color of this portion of the surface is defined by the Lab standard as follows: L: 90.8543 to 94.169; a: 0.83 to -2.8948; b: 72.7371 to 34.3017.

[0008] In some embodiments of the second aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the Lab standard as: L: 90.8543; a: 0.83; b: 72.7371.

[0009] In some embodiments of the second aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the Lab standard as: L: 94.169; a: -2.8948; b: 34.3017.

[0010] In some embodiments of the first and / or second aspects, the cell culture container is a culture dish or a multi-well culture plate.

[0011] In some embodiments of the first and / or second aspects, the substrate is made of paper or rigid material.

[0012] In some embodiments of the first and / or second aspects, the substrate is detachably attached to the bottom of the cell culture container via a groove pre-set in the bottom of the cell culture container.

[0013] In some embodiments of the first and / or second aspects, the substrate is detachably attached to the bottom of the cell culture container by means of a magnetic adsorption element or an adhesive material.

[0014] In some embodiments of the first and / or second aspects, the substrate is detachably attached to the bottom of the cell culture container by means of a snap fastener.

[0015] In some embodiments of the first and / or second aspects, the thickness of the substrate is less than 3 mm.

[0016] Thirdly, this application provides the use of the components described in the first and / or second aspects in bio-optical detection based on firefly luciferase as a reporter gene. Attached Figure Description

[0017] Figure 1 Results of firefly luciferase detection on substrates of 20 colors.

[0018] Figure 2 For example Figure 1 The bar chart shown represents the results of detecting firefly luciferase on 20 different colored substrates; where numbers 1 to 20 correspond to... Figure 1 The paper number in the document. Detailed Implementation

[0019] In cell biology research, various bio-optical detection methods based on chemiluminescence or fluorescence are commonly used. Typically, bio-optical detection is performed in conventional cell culture vessels, which generally have a transparent or fully transparent bottom to facilitate microscopic observation of cells. The inventors of this application hypothesized that this transparency would cause light loss during propagation in bioluminescence, thus affecting the performance of bio-optical tests. Therefore, they researched and explored this issue, leading to the inventions presented in this application.

[0020] For example, common types of luciferase include firefly luciferase, Renilla luciferase, NanoLuc® (NLuc) luciferase, and Gaussia luciferase.

[0021] Firefly luciferase is a 62 kDa monomeric enzyme that, in Mg... 2+ In the presence of ATP, the enzyme binds to the substrate D-luciferin to form an enzyme-luciferin-adenine intermediate, which is oxidized by molecular oxygen to produce high-energy oxidative luciferin. Subsequently, the high-energy electron spontaneously transitions to the ground state, producing a yellow-green photon with an emission wavelength of 550-570 nm. Firefly luciferase is widely used in reporter gene systems. For example, a reporter gene expression plasmid can be constructed by cloning the promoter sequence of the target gene upstream of the coding sequence of firefly luciferase. Transfecting the plasmid into cells allows transcriptional activation of the target gene, and when transcription factors bind to the promoter sequence on the plasmid, initiating the expression of firefly luciferase. By detecting luciferase activity, the transcriptional activation level of the target gene can be analyzed. Firefly luciferase is also widely used in gene regulation research, ATP measurement, drug screening, and signaling pathway analysis. Luciferase-mediated chemiluminescence reactions have advantages such as low background noise and a wide linear range of signal intensity, making them commonly used for high-throughput screening at the cellular level. Multiwell plates with colorless, transparent bottoms are commonly used culture tools for screening.

[0022] In the research and development process of this application, the inventors used firefly luciferase, Renal luciferase, and NanoLuc® (NLuc) luciferase as test substances, and conventional chemiluminescence detection multi-well plates (with white opaque sidewalls and a colorless transparent bottom) as testing tools. In luminescence testing, the transparent bottom of the multi-well plate was shielded with opaque substrates of various colors, and the luminescence intensity was measured and compared. The study found that shielding with opaque substrates of various colors improved luminescence detection, and different colors of shielding substrates produced different effects. Furthermore, it was found that the color of the shielding substrate that achieved the best luminescence detection performance improvement differed among firefly luciferase, Renal luciferase, and NanoLuc® (NLuc) luciferase. This suggests that differences in the optical properties of bioluminescence (such as differences in the maximum emission wavelength of bioluminescence) also affect the optimal color of the shielding substrate, which requires further investigation.

[0023] In a first aspect, this application provides a component for cell culture and bio-optical detection, the component comprising: A cell culture container having a light-transmitting bottom for observing the cultured cells during the cell culture process, and A substrate, detachably attached to the light-transmitting bottom of the cell culture vessel, wherein at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is opaque, and the color of this portion of the surface is defined according to the CMYK standard as follows: C: 0.0389 to 0.0414; M: 0.0794 to 0.1485; Y: 0.3114 to 0.7303; K: 0.0576 to 0.0849.

[0024] In some embodiments of the first aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the CMYK standard as: C: 0.0389; M: 0.0794; Y: 0.3114; K: 0.0576.

[0025] In some embodiments of the first aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the CMYK standard as: C: 0.0414; M: 0.1485; Y: 0.7303; K: 0.0849.

[0026] Secondly, this application provides a component for cell culture and bio-optical detection, the component comprising: A cell culture container having a light-transmitting bottom for observing the cultured cells during the cell culture process, and A substrate, detachably attached to the light-transmitting bottom of the cell culture vessel, wherein at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is opaque, and the color of this portion of the surface is defined by the Lab standard as follows: L: 90.8543 to 94.169; a: 0.83 to -2.8948; b: 72.7371 to 34.3017.

[0027] In some embodiments of the second aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the Lab standard as: L: 90.8543; a: 0.83; b: 72.7371.

[0028] In some embodiments of the second aspect, the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the Lab standard as: L: 94.169; a: -2.8948; b: 34.3017.

[0029] In some embodiments of the first and / or second aspects, the cell culture container may be any conventional culture container in the art, such as a culture dish or a multi-well culture plate, for example, a 4-well culture plate, a 6-well culture plate, a 12-well culture plate, a 24-well culture plate, a 96-well culture plate, a 3.5 cm culture dish, a 6 cm culture dish, a 10 cm culture dish, a 15 cm culture dish, a 24.5 cm culture dish, etc.

[0030] In some embodiments of the first and / or second aspects, the cell culture vessel has a colorless, light-transmitting bottom. A colorless, light-transmitting bottom is also the most common type of conventional culture vessel in the art.

[0031] In some embodiments of the first and / or second aspects, the cell culture vessel has opaque sidewalls, such as white opaque sidewalls. Chemiluminescent detection plates with white opaque sidewalls and colorless, translucent bottoms are commercially available.

[0032] In some embodiments of the first and / or second aspects, when the substrate is attached to the light-transmitting bottom of the cell culture container, at least a portion of the surface of the substrate opposite to the light-transmitting bottom of the cell culture container is opaque; that is, the substrate blocks at least a portion (e.g., 25%, 50%, or 75% (or any percentage between 1% and 100%)) or all of the transparent bottom. With partial blocking, simultaneous microscopic observation and luminescence testing can be achieved; that is, the unblocked portion of the culture container (e.g., a portion of the pores) is used for microscopic observation, and the blocked portion of the culture container (e.g., the remaining pores) is used for luminescence testing. In some embodiments, the substrate may have a matching bottom size to the culture container, and only a portion of the substrate surface is set to be opaque. In other embodiments, the entire surface of the substrate is set to be opaque, but the size is only sufficient to block a portion of the culture container (e.g., a portion of the pores).

[0033] In some embodiments of the first and / or second aspects, the substrate is made of paper or rigid material. There are no particular requirements or limitations on the basic material; any material capable of being opaque and having a specified surface color is acceptable.

[0034] In some embodiments of the first and / or second aspects, the substrate is a paper material.

[0035] In some embodiments of the first aspect and / or the second aspect, the substrate is a rigid material, such as a plastic sheet, PVC sheet, etc.

[0036] In some embodiments of the first and / or second aspects, the substrate is detachably attached to the bottom of the cell culture container via a groove pre-set in the bottom of the cell culture container. In some embodiments of the first and / or second aspects, the substrate is detachably attached to the bottom of the cell culture container by a magnetic adsorption element or an adhesive material. In some embodiments of the first and / or second aspects, the adhesive material is double-sided tape. In some embodiments of the first and / or second aspects, the substrate is detachably attached to the bottom of the cell culture container by a snap-fit. There are no particular limitations on the detachable attachment mechanism in this application; any detachable technology capable of tightly attaching an additional bottom to the cell culture container can be used.

[0037] In some embodiments of the first and / or second aspects, the thickness of the substrate is less than 3 mm.

[0038] In some embodiments of the first and / or second aspects, the thickness of the substrate is less than 2.9 mm, 2.8 mm, 2.7 mm, 2.6 mm, 2.5 mm, 2.4 mm, 2.3 mm, 2.2 mm, 2.1 mm, 2.0 mm, 1.9 mm, 1.8 mm, 1.7 mm, 1.6 mm, 1.5 mm, 1.4 mm, 1.3 mm, 1.2 mm, 1.1 mm, 1.0 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm.

[0039] In some embodiments of the first and / or second aspects, the thickness of the substrate is 2.9 mm, 2.8 mm, 2.7 mm, 2.6 mm, 2.5 mm, 2.4 mm, 2.3 mm, 2.2 mm, 2.1 mm, 2.0 mm, 1.9 mm, 1.8 mm, 1.7 mm, 1.6 mm, 1.5 mm, 1.4 mm, 1.3 mm, 1.2 mm, 1.1 mm, 1.0 mm, 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, or 0.1 mm.

[0040] Thirdly, this application provides the use of the components described in the first and / or second aspects in bio-optical detection based on firefly luciferase as a reporter gene.

[0041] Example

[0042] This application will be described in more detail by way of specific examples. The following embodiments are provided for illustrative purposes only and are not intended to limit this application in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

[0043] Material

[0044] Firefly luciferase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10221-5mg; 96-well transparent-bottom plates were purchased from Corning, catalog number 3903; the Bright-Lumi firefly luciferase reporter gene assay kit was purchased from Beyotime Biotechnology, catalog number RG051M; and Passive Lysis 5X Buffer (PLB) was purchased from Promega, catalog number E1941. The different colored masking substrates were commercially available cardboard; see [reference needed for color information]. Figure 1The CMYK and Lab values ​​of the masked substrate were detected using an X-Rite Exact spectrophotometer, and the results are shown in Table 1.

[0045] Table 1: Figure 1 The CMYK and Lab values ​​of the substrate shown

[0046] method

[0047] Firefly luciferase was dissolved in 1×PLB solution and diluted with PLB solution. 50 μL of enzyme solution was added to both the blank control (bottom unmasked) and the test (bottom masked using substrates of various colors via adhesive). 50 μL of firefly luciferase reporter gene assay reagent was added using an autosampler. Chemiluminescence was detected using a Thermo Fisher multi-plate reader (Fluoroskan Ascent FL), with an integration time of 2 s and a lag time of 2 s after sample addition.

[0048] result

[0049] The effect of different colored substrate masking on the chemiluminescence signal was investigated, and the results are as follows: Figure 1 and Figure 2 As shown, compared to the blank control, the masking of substrates of various colors improved the detection signal. Substrates 3 and 4, which appear yellow, showed the most significant improvement, at 5.077 times and 5.347 times respectively. It is noteworthy that substrate 20 is white. While white is generally considered to have total internal reflection, its improvement in detection signal was weaker than that of substrates 3 and 4, suggesting that the properties of emitted light also affect the improvement effect brought about by substrate masking.

[0050] Furthermore, Renilla luciferase and NanoLuc® (NLuc) luciferase were tested using a similar method. The results showed that masking with substrates of various colors improved the detection signal, but substrates 3 and 4 did not provide the optimal signal enhancement. This further suggests that differences in luminescence properties (e.g., variations in the emitted light wavelength range) also influence the optimal color of the masking substrate, requiring further investigation.

Claims

1. A component for cell culture and bio-optical detection, said component comprising: A cell culture container having a light-transmitting bottom (e.g., a colorless, light-transmitting bottom) and optionally, having opaque sidewalls (e.g., white, opaque sidewalls). A substrate, detachably attached to the light-transmitting bottom of the cell culture vessel, wherein at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is opaque, and the color of this portion of the surface is defined according to the CMYK standard as follows: C: 0.0389 to 0.0414; M: 0.0794 to 0.1485; Y: 0.3114 to 0.7303; K: 0.0576 to 0.0849.

2. The component of claim 1, wherein the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined according to the CMYK standard as: C: 0.0389; M: 0.0794; Y: 0.3114; K: 0.0576.

3. The component of claim 1, wherein the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined according to the CMYK standard as: C：0.0414； M：0.1485； Y: 0.7303; K: 0.0849.

4. A component for cell culture and bio-optical detection, said component comprising: A cell culture container having a light-transmitting bottom (e.g., a colorless, light-transmitting bottom) and optionally, having opaque sidewalls (e.g., white, opaque sidewalls). A substrate, detachably attached to the light-transmitting bottom of the cell culture vessel, wherein at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is opaque, and the color of this portion of the surface is defined by the Lab standard as follows: L: 90.8543 to 94.169; a: 0.83 to -2.8948; b: 72.7371 to 34.3017.

5. The component of claim 4, wherein the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the Lab standard as: L: 90.8543; a: 0.83; b: 72.7371.

6. The component of claim 4, wherein the color of at least a portion of the surface of the substrate opposite the light-transmitting bottom of the cell culture vessel is defined by the Lab standard as: L: 94.169; a: -2.8948; b: 34.3017.

7. The component of any one of claims 1 to 6, wherein the cell culture container is a culture dish or a multi-well culture plate.

8. The component of any one of claims 1 to 6, wherein the substrate is made of paper or rigid material.

9. The component as claimed in any one of claims 1 to 6, wherein, The substrate is detachably attached to the bottom of the cell culture container via a groove pre-set in the bottom of the cell culture container; or The substrate is detachably attached to the bottom of the cell culture container by magnetic adsorption or adhesive material; or The substrate is detachably attached to the bottom of the cell culture container via a snap-fit ​​mechanism.

10. Use of the component of any one of claims 1 to 9 in bio-optical detection based on firefly luciferase as a reporter gene.

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