A culture supplement for enhancing albumin synthesis of liver precursor cells in vitro culture and its use

Abstract

This invention discloses a culture additive for enhancing albumin synthesis in in vitro cultured liver progenitor cells and its application, belonging to the field of cell culture. The culture additive comprises the following components: (a) arvilin or a pharmaceutically acceptable salt thereof, at a concentration of 10–50 μM; (b) a selective HDAC3 inhibitor, at a concentration of 0.1–5 μM; (c) a branched-chain amino acid (BCAA) enhancement solution, with a final concentration 1.5–3 times that of the BCAA concentration in the standard culture medium; and (d) a zinc ion donor, at a concentration of 1–20 μM. This invention upregulates the expression level of the core transcription factor HNF4α of the ALB gene by arvilin, specifically opens the ALB gene locus chromatin by the selective HDAC3 inhibitor, activates the mTORC1 pathway to improve translation efficiency by enhancing BCAAs, and maintains the DNA-binding activity of the HNF4α zinc finger domain by zinc ions. It synergistically enhances the albumin synthesis capacity of liver progenitor cells at four levels: transcriptional activation, epigenetic regulation, translational support, and cofactor supply, while maintaining other hepatocyte functions such as urea synthesis and CYP3A4 enzyme activity.

Description

Technical Field

[0001] This application relates to the field of cell culture and regenerative medicine technology, and more specifically, it relates to a culture additive that enhances albumin synthesis in in vitro cultured liver progenitor cells and its application. Background Technology

[0002] Hepatic progenitor cells (HPCs) are a type of liver progenitor cells with bidirectional differentiation potential, capable of differentiated into mature hepatocytes or bile duct cells in vitro. Obtaining functionally mature in vitro cultured hepatocytes has significant application value in regenerative medicine, drug screening, toxicity evaluation, and the construction of liver disease models. Albumin (ALB), synthesized and secreted by mature hepatocytes, is one of the core indicators for evaluating hepatocyte functional maturity. The albumin secretion level of mature human hepatocytes typically reaches 10–50 μg / 24h / 10 6 Cells, while in existing in vitro culture systems, liver progenitor cells, after maturation induction, only secrete 1-5 μg / 24h / 10 [units of protein]. 6 The number of cells is far lower than that of mature liver cells in vivo, which has become a key technical bottleneck restricting the in vitro application of liver precursor cells.

[0003] Currently, culture systems that promote the differentiation of hepatic progenitor cells into mature hepatocytes mainly rely on classical factor combinations, including cytokines such as hepatocyte growth factor (HGF), oncogene M (OSM), and fibroblast growth factor 2 (FGF2), hormones such as dexamethasone (Dex), and mixtures such as ITS-X (insulin-transferrin-selenoethanol). These factors promote the expression of genes in the direction of liver differentiation by activating corresponding signaling pathways. Patent literature has reported various formulations of hepatic progenitor cell maturation media, such as using DMEM / F12 or IMDM as the basal medium, adding TGF-β inhibitors and γ-secretase inhibitors to relieve differentiation inhibition signals, or using OSM in combination with Dex and ITS-X for induction at the terminal maturation stage. Some literature also introduces non-selective histone deacetylase inhibitors such as sodium butyrate, attempting to further promote liver maturation through epigenetic regulation.

[0004] While the aforementioned culture systems can promote hepatic differentiation of hepatic progenitor cells to some extent, their enhancement of albumin synthesis is limited. This is because these systems are not systematically designed to target the synthetic pathway of albumin, a large secretory protein. Albumin is a large secretory protein composed of 585 amino acids, and its synthesis involves three closely coupled steps: ALB gene transcriptional activation, enhanced polypeptide chain translation efficiency, and endoplasmic reticulum processing and secretion. Existing systems mainly promote overall hepatic differentiation through empirical addition of cytokines, but fail to directly target the activation or upregulation of HNF4α, the core transcription factor of the albumin gene, resulting in low transcriptional-driven efficiency of the ALB gene. Furthermore, the amino acid ratio of conventional culture media is not optimized for albumin translation. Branched-chain amino acids such as leucine can upregulate ribosome translation efficiency by activating the mTORC1 signaling pathway. This mechanism has been reported in primary hepatocytes, but it has not been fully utilized in hepatic progenitor cell maturation culture systems.

[0005] Meanwhile, HNF4α is a C4 type zinc finger protein, and its DNA-binding domain is Zn-dependent. 2+ Maintaining proper folding is crucial, but the zinc ion content in existing culture media is only at trace levels, insufficient to meet the requirements for efficient binding of HNF4α to the ALB promoter. At the epigenetic regulation level, the action sites of existing systems using pan-HDAC inhibitors such as sodium butyrate are imprecise, exhibiting widespread off-target effects that may affect the normal expression of other hepatocyte-related genes. Furthermore, sodium butyrate is cytotoxic at high concentrations, with a narrow safe concentration window, making it difficult to achieve precise regulation of chromatin opening.

[0006] In summary, there is an urgent need in this field for a combination of culture additives with well-defined components, good reproducibility, and targeting the albumin synthesis pathway, which can synergistically enhance the albumin synthesis capacity of hepatic progenitor cells at multiple levels, including transcriptional activation, epigenetic regulation, translational support, and cofactor supply. Summary of the Invention

[0007] To address the aforementioned technical problems, this application provides a culture additive that enhances albumin synthesis in in vitro cultured liver progenitor cells and its application.

[0008] The technical solution adopted in this application is as follows: In a first aspect, this application provides a culture additive that enhances albumin synthesis in in vitro cultured liver progenitor cells, comprising the following components: (a) Alverne or a pharmaceutically acceptable salt thereof, at a concentration of 10–50 μM; (b) Selective inhibitors of HDAC3, at concentrations of 0.1–5 μM; (c) Branched-chain amino acid fortification solution, with a final concentration of 1.5 to 3 times that of the branched-chain amino acid concentration in the standard culture medium; (d) Zinc ion donor, concentration of 1~20 μM.

[0009] Furthermore, the HDAC3 selective inhibitor mentioned above is RGFP966, with a concentration of 0.5~2 μM.

[0010] Furthermore, in the above-mentioned branched-chain amino acid fortification solution, the final concentration of L-leucine is 0.70~0.85 mM, the final concentration of L-isoleucine is 0.35~0.42 mM, and the final concentration of L-valine is 0.70~0.85 mM.

[0011] Furthermore, the zinc ion donor mentioned above is zinc chloride, with a concentration of 3~8 μM.

[0012] Secondly, this application provides a liver progenitor cell maturation culture medium, which comprises a basal culture medium and the above-mentioned culture additives.

[0013] Furthermore, the aforementioned basal culture medium is DMEM / F12 medium, and also contains the following basic factors: hepatocyte growth factor 15~25 ng / mL, oncogene M 5~15 ng / mL, and dexamethasone 0.5~2×10⁻⁶. -8 mol / L, and ITS-X additive 0.5~2 v / v.

[0014] Thirdly, this application provides a method for enhancing albumin synthesis in in vitro cultured liver progenitor cells, comprising the following steps: (1) Seed liver progenitor cells into culture dishes coated with laminin-511 or fragments thereof; (2) The liver progenitor cells were matured using the aforementioned liver progenitor cell maturation medium; (3) Culture for 10 to 14 days, changing the medium every 2 to 3 days during this period.

[0015] Furthermore, the aforementioned liver progenitor cells are derived from directed differentiation of human pluripotent stem cells, in vitro expansion of primary hepatocytes, or hepatic induction of mesenchymal stem cells.

[0016] Furthermore, the aforementioned laminin-511 fragment is iMatrix-511, with a coating concentration of 1–3 μg / cm³. 2 The culture was carried out under conditions of 5-8 v / v% oxygen tension, 37 °C, and 5 v / v% CO2.

[0017] Fourthly, this application also provides the use of the above-mentioned culture additive in the preparation of a cell culture medium for enhancing albumin synthesis in liver progenitor cells.

[0018] In summary, this application has the following beneficial effects: The culture additive combination of this invention upregulates the expression level of HNF4α, the core transcription factor of the ALB gene, through alvirine, while simultaneously, in combination with a selective HDAC3 inhibitor, specifically increases the histone acetylation level in the ALB gene promoter region, making the chromatin structure more open and facilitating HNF4α access and binding to the ALB promoter. This dual strategy of transcriptional activation and epigenetic opening works synergistically to significantly enhance the albumin synthesis and secretion capacity of cultured liver progenitor cells during maturation.

[0019] The branched-chain amino acid fortification solution in the culture additive combination of this invention activates the mTORC1 signaling pathway by increasing the concentrations of L-leucine, L-isoleucine, and L-valine, thereby promoting ribosome translation efficiency and enhancing the synthesis rate of albumin polypeptide chains. Quantitative supplementation of Zn using zinc ion donors is also included. 2+ This maintains the correct folding of the HNF4α C4 type zinc finger domain, enhances its binding affinity to the ALB promoter, and further amplifies the transcriptional activation effect.

[0020] The combination of culture additives in this invention enhances albumin synthesis while maintaining other hepatocyte function indicators such as urea synthesis, CYP3A4 enzyme activity, and glycogen storage without decreasing. This demonstrates that the effects of this invention have a relatively specific effect on the albumin synthesis pathway, rather than a non-specific global enhancement of hepatocyte function. Detailed Implementation

[0021] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The key reagents and raw materials used in the embodiments of this application are shown in Table 1: Table 1. Sources of Reagent Raw Materials Reagents and biological materials whose specific sources are not specified in this application are all commercially available products in the field, or can be prepared by conventional methods known in the field.

[0023] Hepatic progenitor cell source: Using a published method (Stephens et al., Nature Protocols, 2018), H9 human embryonic stem cells (hESC, WA09, WiCell) were used to obtain hepatic progenitor cells through the following steps: The first stage was definitive endoderm induction, cultured for 3 days in RPMI 1640 / B27 medium containing Activin A (100 ng / mL) and Wnt3a (25 ng / mL); the second stage was hepatic progenitor induction, cultured for 5 days in DMEM / F12 / B27 medium containing BMP4 (50 ng / mL) and bFGF (20 ng / mL). The obtained hepatic progenitor cells expressed AFP and CK19 and exhibited a typical hepatic progenitor cell phenotype.

[0024] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0025] Example 1 This embodiment provides a method for preparing culture additives and inducing liver progenitor cell maturation. I. Preparation of stock solutions for each component: (1) Alverine stock solution: Weigh Alverine (CAS: 5560-59-8) standard and dissolve it in DMSO to prepare a 50 mM stock solution, and store it in aliquots at -20 ℃; (2) RGFP966 stock solution: Weigh RGFP966 standard and dissolve it in DMSO to prepare a 10 mM stock solution, and store it in aliquots at -20 ℃. (3) BCAA enhancement solution: Weigh 0.78 mmol of L-leucine, 0.38 mmol of L-isoleucine and 0.78 mmol of L-valine respectively, dissolve them in ultrapure water, adjust the pH to 7.0~7.4 with 1 M NaOH, make up to 100 mL, filter through a 0.22 μm filter membrane for sterilization, and store at 4 ℃; (4) ZnCl2 stock solution: Weigh ZnCl2 and dissolve it in ultrapure water to prepare a 10 mM stock solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4 ℃.

[0026] II. Induction of Hepatic Progenitor Cell Maturation Hepatic progenitor cells were 5 × 10 4 cells / cm 2 The density of inoculation was 2 μg / cm² in iMatrix-511 coated cells. 2The culture medium was prepared in 24-well plates. The maturation medium consisted of DMEM / F12 basal medium supplemented with 25 μM Alverne, 1 μM RGFP966, BCAA enhancement solution (final concentration twice that of standard DMEM / F12 BCAA), 25 μM ZnCl, and HGF 20 ng / mL, OSM 10 ng / mL, and dexamethasone 1×10⁻⁶. -8 mol / L, ITS-X 1 v / v%. Culture conditions were 37 ℃, 5 v / v% O2, 5 v / v% CO2, 90 v / v% N2. Culture time was 14 days, with medium changed every 2 days.

[0027] Example 2 The culture system in this embodiment is the same as that in Example 1, except that the concentration of Alverne is adjusted to 10 μM, while the concentrations of other components remain unchanged (RGFP966 1 μM, BCAA fortified 2 times, ZnCl2 5 μM). The culture conditions are the same as in Example 1.

[0028] Example 3 The culture system in this embodiment is the same as that in Example 1, except that the concentration of Alverne is adjusted to 50 μM, while the concentrations of other components remain unchanged. The culture conditions are the same as in Example 1.

[0029] Example 4 The culture system in this embodiment is the same as that in Example 1, except that the concentration of RGFP966 is adjusted to 0.5 μM, while the concentrations of other components remain unchanged (Alverne 25 μM, BCAA fortified 2 times, ZnCl 25 μM). The culture conditions are the same as in Example 1.

[0030] Example 5 The culture system in this embodiment is the same as that in Example 1, except that the ZnCl2 concentration is adjusted to 3 μM, while the concentrations of other components remain unchanged (Alverne 25 μM, RGFP966 1 μM, BCAA fortification 2-fold). The culture conditions are the same as in Example 1.

[0031] Example 6 The culture system in this embodiment is the same as that in Example 1, except that the final concentration of the BCAA enhancement solution is adjusted to 1.5 times the concentration of the DMEM / F12 standard BCAA, while the concentrations of other components remain unchanged (Alverne 25 μM, RGFP966 1 μM, ZnCl 25 μM). The culture conditions are the same as in Example 1.

[0032] Comparative Example 1 The culture system for this comparative example is the same as that in Example 1, except that it does not contain Alvirin, while other components remain unchanged (RGFP966 1 μM, BCAA fortification 2-fold, ZnCl2 5 μM, HGF 20 ng / mL, OSM 10 ng / mL, dexamethasone 1×10⁻⁶). - 8 (mol / L, ITS-X 1 v / v%). The culture conditions were the same as in Example 1.

[0033] Comparative Example 2 The culture system for this comparative example is the same as that in Example 1, except that it does not contain RGFP966, while other components remain unchanged (Alverne 25 μM, BCAA fortified 2 times, ZnCl 25 μM). The culture conditions are the same as in Example 1.

[0034] Comparative Example 3 The culture system for this comparative example was the same as that in Example 1, except that it did not contain BCAA enhancement solution. The BCAA concentration was maintained at the standard DMEM / F12 concentration (L-leucine 0.39 mM, L-isoleucine 0.19 mM, L-valine 0.39 mM), and other components remained unchanged (alvirine 25 μM, RGFP966 1 μM, ZnCl 25 μM). The culture conditions were the same as in Example 1.

[0035] Comparative Example 4 The culture system for this comparative example is the same as that in Example 1, except that no additional ZnCl2 is added. The zinc ion concentration comes only from trace amounts of zinc in the DMEM / F12 basal medium and ITS-X. Other components remain unchanged (Alverline 25 μM, RGFP966 1 μM, BCAA fortified 2 times). The culture conditions are the same as in Example 1.

[0036] Comparative Example 5 The culture system for this comparative example is the same as that in Example 1, except that RGFP966 is replaced with 0.5 mM sodium butyrate, while other components remain unchanged (25 μM Alverne, 2-fold BCAA fortification, 25 μM ZnCl). The culture conditions are the same as in Example 1.

[0037] Comparative Example 6 This comparative example uses a classic liver progenitor cell maturation protocol from existing technologies to verify the technical effectiveness of the culture additive combination of this invention compared to existing technologies. The culture system was: DMEM / F12 basal medium supplemented with HGF 20 ng / mL, OSM 10 ng / mL, and dexamethasone 1×10⁻⁶. -8mol / L, ITS-X 1 v / v%, free of Alverne, RGFP966, BCAA enhancement solution, and ZnCl2. Cells were seeded in iMatrix-511 coated cells (2 μg / cm²). 2 In a 24-well plate, the inoculation density is 5 × 10⁻⁶. 4 cells / cm 2 The culture conditions were 37 ℃, 5 v / v% O2, 5 v / v% CO2, and 90 v / v% N2. The culture time was 14 days, with the medium changed every 2 days.

[0038] Performance testing I. Albumin secretion and cell viability Albumin secretion assay: Culture supernatant from each group was collected every 2 days and stored at -80 ℃ for later use. Quantitative detection was performed using a human albumin ELISA kit (Bethyl Laboratory) according to the manufacturer's instructions. A standard curve was constructed using known concentrations of human albumin standards. Results were expressed as μg / 24h / 10 6 Cellular representation.

[0039] Cell viability detection method: After 14 days of culture, the cell viability of each group was detected by CCK-8 assay. 10 μL of CCK-8 reagent was added to each well, and after incubation at 37 ℃ for 2 h, the absorbance at 450 nm was measured using an ELISA reader. The relative viability of each group was calculated with the absorbance of the group in Example 1 as the baseline (100%).

[0040] The test results are as follows: Table 2. Results of albumin secretion and cell viability assay As shown in Table 2, the albumin secretion levels in Examples 1-6 were significantly higher than those in the classic system of Comparative Example 6 (2.8 μg / 24h / 10). 6 (cells), Example 1 showed the highest albumin secretion under optimal conditions, reaching 35.8 μg / 24h / 10 6 The cell count was approximately 12.8 times that of the classic system. Examples 2-6 verified the effects of acetaminophen, RGFP966, ZnCl2, and BCAA enhancement at the endpoints of the concentration ranges defined in the claims, with albumin secretion reaching 28.5-34.2 μg / 24h / 10. 6 The cells showed significantly better performance than the classic system, demonstrating that the numerical range defined in the claims is reasonable and feasible. The cell viability in each embodiment was between 99% and 101%, indicating that the culture additive combination of the present invention has good biosafety within the concentration range defined in the claims.

[0041] Comparative Examples 1-4 were performed by removing each component of the culture additive combination of the present invention one by one. The results showed that the albumin secretion level decreased significantly after removing any component, with the removal of alverlin having the most significant effect (from 35.8 to 8.5 μg / 24h / 10). 6 (The data from the cells indicates that the upregulation of HNF4α expression by Alvirin, as an upregulator, is the core driving factor of this treatment.) The removal effect of each component showed a progressive decrease, demonstrating a synergistic enhancement effect among the four components.

[0042] Comparative Example 5 used sodium butyrate instead of RGFP966, and the albumin secretion level was 18.2 μg / 24h / 10. 6 The cell count was significantly lower than that of the RGFP966 regimen (35.8 μg / 24h / 10 cells). 6 The cells were isolated and the cell viability dropped to 92%, which verified the dual advantages of selective HDAC3 inhibition over non-selective HDAC inhibition in terms of technical efficacy and safety.

[0043] II. Verification of Hepatocyte Comprehensive Function, Transcription Level, and Translation Efficiency This group of experiments mainly compares the various supporting indicators of Example 1 (optimal group) and Comparative Example 6 (classical system).

[0044] Methods for detecting comprehensive hepatocyte function: Urea synthesis was detected using a urea nitrogen assay kit. After stimulating the culture medium with 1 mM NH4Cl for 24 h, the supernatant was collected for quantification. CYP3A4 activity was detected by a fluorescence substrate method. BFC (5 μM) was used as the substrate, and the amount of fluorescent product generated was detected after induction with 25 μM rifampicin for 24 h. Glycogen storage was performed by PAS staining, and the staining area ratio was quantified using ImageJ software. LDL uptake was assessed by incubating with Dil-LDL (10 μg / mL) for 4 h and then detecting intracellular fluorescence intensity. Tight junction integrity was determined by immunofluorescence staining to detect the continuous distribution of ZO-1 protein.

[0045] ALB gene transcription level detection method: Cells were collected after 14 days of culture, and total RNA was extracted using TRIzol reagent. After quantification with NanoDrop, it was reverse transcribed into cDNA. The relative expression levels of ALB mRNA and HNF4α mRNA were detected by qRT-PCR using GAPDH as an internal reference gene. - The ΔΔCt method is used to calculate relative multiples.

[0046] Translation efficiency was assessed using the SUnSET (Surface Sensing of Translation) method to detect the rate of nascent protein synthesis. On day 14 of culture, puromycin (1 μg / mL) was added to the culture medium of each group and incubated for 30 min. After cell collection, Western blotting was performed to detect the puromycin signal incorporating nascent polypeptide chains, with the total protein loading amount serving as an internal control. Simultaneously, the phosphorylation levels of mTORC1 downstream targets p-S6 (Ser235 / 236) and p-4E-BP1 (Thr37 / 46) were detected, and the grayscale values ​​were quantified using ImageJ. The results are shown in Table 3. Table 3. Results of Supporting Indicators As shown in Table 3, in terms of comprehensive hepatocyte function, the urea synthesis, CYP3A4 enzyme activity, glycogen storage, LDL uptake and tight junction integrity of Example 1 were not lower than those of Comparative Example 6, and some indicators showed a slight upward trend, proving that the combination of culture additives of the present invention enhanced albumin synthesis without negatively affecting other core hepatocyte functions.

[0047] At the transcriptional level, the ALB mRNA expression level in Example 1 reached 15.3 times that of Comparative Example 6, and the HNF4α mRNA was simultaneously upregulated to 4.2 times, verifying the mechanism by which Alvirin upregulates HNF4α expression and drives ALB gene transcription.

[0048] Regarding translation efficiency, the amount of puromycin incorporated in Example 1 was 2.5 times that of Comparative Example 6, and the phosphorylation levels of p-S6 and p-4E-BP1 were increased by 2.3 times and 2.0 times, respectively, verifying the mechanism by which BCAA enhancement improves ribosomal translation efficiency by activating the mTORC1 pathway.

[0049] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A culture additive that enhances albumin synthesis in in vitro cultured liver progenitor cells, characterized in that, It contains the following components: (a) Alverne or a pharmaceutically acceptable salt thereof, at a concentration of 10–50 μM; (b) Selective inhibitors of HDAC3, at concentrations of 0.1–5 μM; (c) Branched-chain amino acid fortification solution, with a final concentration of 1.5 to 3 times that of the branched-chain amino acid concentration in the standard culture medium; (d) Zinc ion donor, concentration of 1~20 μM.

2. The culture additive according to claim 1, characterized in that, The HDAC3 selective inhibitor is RGFP966, with a concentration of 0.5–2 μM.

3. The culture additive according to claim 1, characterized in that, In the branched-chain amino acid fortification solution, the final concentration of L-leucine is 0.70~0.85 mM, the final concentration of L-isoleucine is 0.35~0.42 mM, and the final concentration of L-valine is 0.70~0.85 mM.

4. The culture additive according to claim 1, characterized in that, The zinc ion donor is zinc chloride with a concentration of 3-8 μM.

5. A liver progenitor cell maturation culture medium, characterized in that, It comprises a basal culture medium and the culture additives as described in any one of claims 1 to 4.

6. The liver progenitor cell maturation culture medium according to claim 5, characterized in that, The basal culture medium is DMEM / F12 medium, and also contains the following basic factors: hepatocyte growth factor 15~25 ng / mL, oncogene M 5~15 ng / mL, dexamethasone 0.5~2×10⁻⁶. -8 mol / L, and ITS-X additive 0.5~2 v / v.

7. A culture method for enhancing albumin synthesis in in vitro cultured liver progenitor cells, characterized in that, Includes the following steps: (1) Seed liver progenitor cells into culture dishes coated with laminin-511 or fragments thereof; (2) The liver progenitor cells were matured using the liver progenitor cell maturation medium as described in claim 5 or 6; (3) Culture for 10 to 14 days, changing the medium every 2 to 3 days during this period.

8. The cultivation method according to claim 7, characterized in that, The liver progenitor cells are derived from directed differentiation of human pluripotent stem cells, in vitro expansion of primary hepatocytes, or hepatic induction of mesenchymal stem cells.

9. The cultivation method according to claim 7, characterized in that, The laminin-511 fragment is iMatrix-511, with a coating concentration of 1~3 μg / cm³. 2 The culture was carried out under conditions of 5-8 v / v% oxygen tension, 37 °C, and 5 v / v% CO2.

10. The use of a culture additive as described in any one of claims 1 to 4 in the preparation of a cell culture medium for enhancing albumin synthesis in liver progenitor cells.

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