Cell calcification method and screening method using the same

Abstract

A method for calcifying cells, comprising suspending mesenchymal stem cells in 85-100% serum, allowing the suspended mesenchymal stem cells to stand at 4-10°C for at least 4 days, seeding the mesenchymal stem cells after standing in a culture vessel, and culturing them in a serum-free basal medium to calcify them.

Description

[Technical Field]

[0001] The present invention relates to a method for calcifying cells and a screening method using the method for calcifying cells. [Background technology]

[0002] In recent years, there has been a demand for the development of drugs related to calcification, such as calcification promoters that have therapeutic effects on osteoporosis, and calcification inhibitors that suppress cartilage calcification and stone formation in the body.

[0003] For example, Patent Document 1 provides a calcium calcification inhibitor that inhibits cartilage calcification and stone formation in the body, based on the mechanism of calcium calcification in the human body. Furthermore, Patent Document 2 provides an osteoblast calcification promoter that can be used as a therapeutic agent for osteoporosis.

[0004] However, in order to test (screen) the effectiveness of the developed calcification promoters and calcification inhibitors, for example, to prepare a test environment for calcification using stem cells, it takes time to culture the stem cells, induce bone differentiation, and cause calcification, which means there are many work steps and it takes a long time. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2006-104152 [Patent Document 2] Japanese Patent Application Publication No. 05-000965 Summary of the Invention [Problem to be solved by the invention]

[0006] Osteoblasts are thought to differentiate from mesenchymal stem cells, and in bone and dental tissues, osteoblasts differentiate into osteocytes, which are responsible for mineralization. Normally, mineralization by osteocytes is regulated so that it occurs where it is needed in the body. However, some abnormality can cause calcification in various parts of the body (except bones and teeth), leading to movement and physiological problems. This phenomenon is called ectopic calcification (ossification), and is known to occur when hydroxyapatite crystals are deposited on collagen fibers in soft tissues, but the mechanism behind this remains unknown.

[0007] In light of the above circumstances, the present inventors conducted further research and discovered a new method for inducing calcification by using 85 to 100% serum in cell culture, which has a different mechanism of action from the calcification that occurs when mesenchymal stem cells (MSCs) are induced to differentiate into osteoblasts, thereby leading to the present invention. Specifically, we found that calcification occurs when cells are cultured in 85-100% serum for a certain period of time. Serum such as fetal bovine serum (FBS), adult bovine serum (BS), and human adult serum can be used depending on the cell type. We also found that a calcium concentration of 100 mg / L or higher is important for efficient calcification.

[0008] The present invention has been made in consideration of the above-mentioned circumstances, and aims to provide a method for calcifying cells, which is capable of causing calcification at an early stage by cell culture without using a differentiation-inducing medium or the like. [Means for solving the problem]

[0009] The present invention has been made based on the above findings and aims to advantageously solve the above problems. A first aspect of the present invention is: Mesenchymal stem cells are suspended in 85-100% serum. The suspended mesenchymal stem cells are left standing at 4-10°C for at least 4 days. After the incubation, the mesenchymal stem cells are seeded into a culture vessel. A method for calcifying cells by culturing them in a serum-free basal medium; is. According to such a method for calcifying cells, calcification can be caused to occur early by cell culture without using a differentiation-inducing medium or the like.

[0010] In addition, a second aspect of the present invention is a method for calcifying cells, comprising culturing the cells in 85 to 100% serum for at least 4 days, thereby calcifying the cells; is. According to such a method for calcifying cells, calcification can be caused to occur early by cell culture without using a differentiation-inducing medium or the like.

[0011] In the second aspect, the serum is of bovine origin; The cells Mesenchymal stem cells, dental pulp cells, periodontal ligament cells, selected from the group consisting of vascular endothelial cells, epidermal keratinocytes, corneal cells, chondrocytes, hair papilla cells, cell lines, and cancer cells; the cell line is HEC293, L929, Balb / 3T3, MC3T3-E1, or Vero cell; The cancer cells may be A549, THP-1, BRL-2H3, or P3U1. Such a specific combination of serum and cells can induce calcification more effectively.

[0012] In the second aspect, the serum is of human origin; The cells may be mesenchymal stem cells or epidermal keratinocytes. Such a specific combination of serum and cells can induce calcification more effectively.

[0013] In the second aspect, Calcium may be added to the serum so that the concentration is 100 mg / L or more. By doing so, calcification can be more efficiently caused.

[0014] A third aspect of the present invention is a screening method for screening calcification inhibitors using cells calcified by the cell calcification method according to the first or second aspect. According to such a screening method, candidate drugs can be screened using the calcification with extremely high efficiency. [Effects of the Invention]

[0015] According to the present invention, calcification can be caused to occur early by cell culture without using a differentiation-inducing medium or the like. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows the results of Example 1. [Figure 2] FIG. 2 shows the results of Example 2. [Figure 3] FIG. 3 shows the results of Example 4. [Figure 4] FIG. 4 is a table showing the results of Example 5. [Figure 5] FIG. 5 is a table showing the results of Example 6. [Figure 6] FIG. 6 is a table showing the results of Example 7. [Figure 7] FIG. 7 shows the results of Example 8. [Figure 8] FIG. 8 shows the results of Example 9. [Figure 9] FIG. 9 shows the results of Example 10. DETAILED DESCRIPTION OF THE INVENTION

[0017] Hereinafter, embodiments of the present invention will be described in detail. The method for calcifying cells according to the first aspect of the present invention comprises: Mesenchymal stem cells are suspended in 85-100% serum. The suspended mesenchymal stem cells are left standing at 4-10°C for at least 4 days. After the incubation, the mesenchymal stem cells are seeded into a culture vessel. The method is characterized in that the cells are cultured in a serum-free basal medium to calcify the cells, and Furthermore, the method for calcifying cells according to the second aspect of the present invention comprises: The method is characterized in that the cells are calcified by a culture step in which the cells are cultured in 85 to 100% serum for at least 4 days. Furthermore, the screening method according to the third aspect of the present invention is characterized in that it is a screening method for screening calcification inhibitors using cells calcified by the cell calcification method described in the first or second aspect above.

[0018] The inventors have newly discovered that calcification occurs when low-temperature-treated MSCs are cultured in 85-100% FBS for a predetermined period of time. Furthermore, they have newly discovered that calcification occurs when cells are cultured in 85-100% FBS under predetermined conditions, even without low-temperature treatment.

[0019] In the cell calcification method according to the first aspect of the present invention, mesenchymal stem cells that can be used include, for example, bone marrow-derived mesenchymal stem cells, adipose-derived stem cells (ASCs), peripheral blood-derived mesenchymal stem cells, umbilical cord Wharton's Jelly-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, and dental pulp-derived mesenchymal stem cells.

[0020] In the first embodiment, the serum used to suspend the cells may be fetal bovine serum (FBS), adult bovine serum (BS), human adult serum, or the like.

[0021] In the first aspect, the basal medium may be IMDM, DMEM, or α-MEM, but is not limited to these, and any commercially available basal medium suitable for culturing mesenchymal stem cells may be used.

[0022] In the first embodiment, the low-temperature treatment in which the mixture is left standing at 4 to 10° C. is carried out for at least 4 days, preferably 4 to 5 days, and more preferably 4 days.

[0023] In the first aspect, the concentration of cells suspended in 85 to 100% serum during low-temperature treatment is 1 × 10 5 cells / ml ~ 1 × 10 7 cells / ml is preferred. Most preferred is 1.5 x 10 6 Suspend the cells in 1.5 ml of 85–100% serum. In the first aspect, the cell concentration when seeded onto a culture vessel such as a plate during culture after low-temperature treatment is 2.5 × 10 4 cells / ml ~ 10 × 10 4 cells / ml is preferred. Most preferably, 5 x 10 4 cells / well. If serum at a concentration other than 100% is used, dilute it appropriately with basal medium or the like.

[0024] In the cell calcification method according to the second embodiment of the present invention, the cells are cultured in 85 to 100% serum for at least 4 days, but can be cultured for up to 14 days.

[0025] In the second aspect, the cell concentration in the culture step of culturing for at least 4 days in 85 to 100% serum is 2.5 × 10 4 cells / ml ~ 10 × 10 4 cells / ml is preferred. Most preferably, 5 x 10 4 Cells are cultured at a concentration of 100 cells / well in 85-100% serum. If serum at a concentration other than 100% is used, dilute it appropriately with basal medium or the like.

[0026] In the second aspect, the serum used in the culture step may be, but is not limited to, fetal bovine serum (FBS), adult bovine serum (BS), human adult serum, etc., depending on the type of cells to be cultured.

[0027] For example, if the serum is of bovine origin, the cells may be Suitable cells include mesenchymal stem cells, dental pulp cells, periodontal ligament cells, vascular endothelial cells, epidermal keratinocytes, corneal cells, chondrocytes, hair papilla cells, established cell lines, and cancer cells, and examples of such cells include, but are not limited to, HEC293, L929, Balb / 3T3, MC3T3-E1, or Vero cells, and cancer cells include, but are not limited to, HA549, THP-1, BRL-2H3, or P3U1.

[0028] For example, when the serum is derived from a human, the cells are suitably, but not limited to, mesenchymal stem cells or epidermal keratinocytes.

[0029] In the second aspect, calcification can be more efficiently induced by adding calcium to serum at a concentration of 100 mg / L or more. Calcium can be added up to a maximum of 250 mg / L, preferably 100 to 150 mg / L.

[0030] In the screening method according to the third aspect of the present invention, candidate drugs for calcification inhibitors and calcification promoters can be screened using cellular calcification produced by the calcification method according to the first or second aspect, but the candidate drugs are not limited to those for calcification inhibition and calcification promotion, and any candidate drugs related to calcification can be used. [Example]

[0031] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0032] Example 1: Calcification of adipose-derived stem cells (ASCs) by serum culture ASCs (Lonza Co., Ltd., product number: PT-5006) were cultured in a T-75 flask in BSCM-PL1 medium (Bio Mirai Kobo Co., Ltd., product number: BMK-S002) supplemented with 2% FBS (Cytiva, product number: SH30910) until they reached 80-90% confluence. Afterwards, they were detached with trypsin-EDTA (Nacalai Tesque, product number: 32777-44), suspended in the above BSCM-PL1 medium, and plated in 0.5 mL (5 × 10 4 The next day, when the cells reached 100% confluence, the medium was replaced with 100% serum (fetal bovine serum (FBS) / adult bovine serum (BS) / human adult serum) (medium exchange) and the cells were cultured for 10 days. The sera were FBS (Cytiva, product number SH30910), BS (Kohjin Bio, product number 12183610), and human adult serum (Sigma-Aldrich, product number B9433). On days 3, 7, and 10, the tissues were stained with Alizarin Red S staining kit (Bio Mirai Kobo, product number BMK-R009) and examined microscopically for calcification. The results are shown in Figure 1 (magnified micrograph (x64)). As shown in Figure 1, FBS and BS caused calcification from day 3 of culture. In contrast, no calcification was observed in adult human serum even on day 10 (photo not shown).

[0033] (Example 2) Calcification of various cells by serum culture Next, the calcification of the following cells was observed using the above three types of sera: Bone marrow-derived MSCs, Wharton's Jelly-derived mesenchymal stem cells (isolated and cultured from donated human umbilical cords using the method described in the "Mesenchymal Stem Cell Handbook" (Information Organization)), Dental pulp cells, periodontal ligament cells (isolated and cultured from donated human teeth using the method described in the "Mesenchymal Stem Cell Handbook" (Information Organization)), Vascular endothelial cells (HUVEC) (Takara Bio Inc., product number: C-12200), epidermal keratinocytes (Takara Bio Inc., product number: C-12006), chondrocytes (Takara Bio Inc., product number: C-12710), dermal papilla cells (Takara Bio Inc., product number: C-12071), Cell lines: HEC293, L929, Balb / 3T3, MC3T3-E1, Vero cells, corneal cells (SIRC) (obtained from JCRB Cell Bank / RIKEN BRC Cell Bank), Cancer cells: A549, THP-1, BRL-2H3, P3U1 (obtained from JCRB Cell Bank / CELL BANK of RIKEN BRC).

[0034] The above cells were cultured in a commercially available medium suitable for each cell type in the same manner as in Example 1, except that they were cultured until confluent. On days 7 to 11, the cells were stained with Alizarin Red S staining kit and observed under a microscope for the presence or absence of calcification. The results are shown in Table 1 and Figure 2 (microscopic magnification (x64)). As shown in Table 1 and Figure 2, with FBS and BS, calcification was observed in all cells used in the test, but with human adult serum, calcification was observed only in Wharton's Jelly-derived mesenchymal stem cells and epidermal keratinocytes. In Table 1, "◯" indicates that 50% or more of the well bottom area was stained, "△" indicates that 1% to less than 50% of the well bottom area was stained, "×" indicates that there was no staining at all, and "-" indicates that no data was available.

[0035] [Table 1]

[0036] (Example 3) Measurement of serum calcium concentration Since calcium concentration is considered to be one of the important conditions for calcification, the calcium concentrations of the following FBS, BS, and human adult serum, including the serum used in Examples 1 and 2, were measured using the Metalloassay Calcium Measurement LS Kit (Metallogenics, Inc., product number: CA30M). FBS (Cytiva, product number SH30910, Lot. AG29485626 and Lot. AE24573269) BS (Kohjin Bio, product number: 12183610; Sigma Aldrich, product number: B9433) Human adult serum (Sigma-Aldrich, product number B9433; Kohjin Bio, product number 12181201, lot 20952040 and lot 23080478-1) Table 2 shows the mean values of the measurement results for FBS, BS, and human adult serum.

[0037] [Table 2]

[0038] Regarding the results in Table 2, since the data sheet for FBS (Cytiva, product number: SH30910) states the calcium concentration as 139 mg / L, it is believed that the above method can accurately measure calcium concentrations in BS and human adult serum. Furthermore, the results in Table 2 show that the calcium concentration in human adult serum is lower than that in FBS and BS.

[0039] (Example 4) Calcification by culture with adult human serum supplemented with calcium chloride Therefore, we observed calcification in cultures containing human adult serum with an increased calcium concentration. Specifically, the ASCs and periodontal ligament cells used in Examples 1 and 2 were cultured for 11 days in human adult serum (the same serum as in Example 1) supplemented with calcium chloride (Nacalai Tesque, product number: 06900-14) at 100 mg / L, and as in Example 1, stained with an Alizarin Red S staining kit and observed under a microscope for the presence or absence of calcification. The results are shown in Figure 3 (magnified micrograph (x64)). When cultured in human adult serum supplemented with calcium chloride at 100 mg / L, mineralization was observed in both ASCs and periodontal ligament cells. The results of Example 4 show that calcification is effectively promoted by increasing the calcium concentration in serum to 100 mg / L or more.

[0040] (Example 5) ASC mRNA gene expression (1) RNA was extracted from the lysates of ASCs on days 3, 5, and 7 of culture in Example 1 using Maxwell (registered trademark) RSC simplyRNA Cells Kit (Promega Corporation, product number: AS1340). Using the extracted RNA as a template and the TaqMan primers / probes listed in Table 3, qPCR (ThermoFisher Scientific, Step One PLUS PCR system) was performed.

[0041] [Table 3]

[0042] The results are shown in Figure 4. In the control osteoblast differentiation medium (OBM), the expression of osteoblast-related genes ALP, type I collagen, osteocalcin, Runx2, and Sp7 mRNA increased. On the other hand, in 100% FBS, 100% BS, and 100% human adult serum, the expression of all of the above genes did not increase as in OBM, but the expression of some genes did increase.

[0043] (Example 6) mRNA gene expression of Wharton's Jelly-derived mesenchymal stem cells RNA was extracted from the lysate of Wharton's Jelly-derived mesenchymal stem cells on days 3, 5, and 7 of culture in Example 2 using the Maxwell (registered trademark) RSC simplyRNA Cells Kit in the same manner as in Example 5. The results are shown in Figure 5. In the control OBM, osteocalcin and Runx 2 mRNA expression was increased, whereas in the 100% FBS, 100% BS, and 100% human adult serum, no increase in gene expression was observed.

[0044] (Example 7) ASC mRNA gene expression (2) Except for using a different lot of ASCs from those used in Example 4, ASCs were cultured in 100% human adult serum and human adult serum with an elevated calcium concentration, as in Example 4. RNA was extracted from lysates on days 1, 3, and 7 of culture using the Maxwell® RSC simplyRNA Cells Kit in the same manner as in Example 5. The results are shown in Figure 6. In the control OBM, osteocalcin and ALP mRNA expression increased significantly, whereas in the 100% human adult serum and calcium-supplemented human adult serum, no increase in either gene expression was observed over time. From the results of Examples 5 to 7, it is believed that the mechanism of calcification caused by 100% serum is different from that of calcification caused by bone differentiation induction using OBM.

[0045] (Example 8) Calcification by culture after low temperature treatment 1.5 × 10 cells were cultured in the same manner as in Example 1 until they reached 80 to 90% confluence. 6 ASC cells were suspended in 1.5 ml of 100% FBS and placed in a refrigerator (4-10°C) for 2-4 days for low-temperature treatment. After that, 5 × 10 cells were plated onto a 48-well plate. 4Cells were seeded at 1000 cells / well, and after 2 hours the medium was replaced with serum-free α-MEM and cultured for 7 days. The medium was replaced every 3 days during the 7-day culture. Alizarin Red staining was performed on days 1, 3, and 7 of culture in α-MEM. The results are shown in Figure 7. Cells exposed to low temperature for 4 days calcified from the first day of culture, but cells exposed to low temperature for 2 days (photo not included) and 3 days did not calcify even on day 7 of culture. Even when cells were suspended in a medium other than serum (such as osteoblast differentiation-inducing medium) and subjected to low-temperature treatment, calcification did not occur when the cells were subsequently cultured in a serum-free basal medium (data not shown).

[0046] Example 9 In Example 8, mRNA was extracted from the lysate of cells sampled on days 1, 3, and 7 of culture using α-MEM, as in Example 5, and qPCR was performed. The results are shown in Figure 8. As a control, expression was measured in cells cultured only in growth medium (α-MEM). As shown in Figure 8, no specific osteoblast-related gene expression trend was observed. This suggests that the mechanism of mineralization by 100% serum is different from that of mineralization by osteogenic differentiation induction using OBM.

[0047] (Example 10) Difference in calcification depending on FBS concentration ASCs were suspended in a serum-free medium for mesenchymal stem cells containing 2% FBS (Fukoku Co., Ltd., product number: FKCM301T) at 1 × 10 5 The cells were adjusted to a concentration of 100 cells / mL and seeded at 500 μL / well in a 48-well plate. Culture was continued until 100% confluent, and the medium was replaced with 100% FBS, 90% FBS, 85% FBS, 75% FBS, or 50% FBS at 300 μL / well. FBS (Cytiva, product number: SH30910) was used, and saline (Otsuka Pharmaceutical, product number: 1326) was used for dilution. On the fourth day of culture, the cells were stained with Alizarin Red in the same manner as in Example 1, then extracted with 5% formic acid, and the OD at 450 nm / 630 nm was measured using a Nivo multimode plate reader (Revvity). The results are shown in Figure 9. The degree of calcification was highest with 100% FBS, and high calcification was observed up to 85% FBS. The degree of calcification decreased with 75% FBS, and no calcification occurred with 50% FBS. This indicates that a high concentration of FBS is important for calcification.

[0048] (Example 11) Screening of calcification inhibitors 1 To investigate whether it is possible to screen for calcification inhibitors using cell calcification in 100% serum culture, the calcification inhibitors listed in Nos. 1 to 3 in Table 4 were prepared at 10 mM, 1 mM, and 100 μM in α-MEM medium containing 2% FBS (Nacalai Tesque, Inc., product number: 21445).

[0049] [Table 4]

[0050] Next, ASCs were suspended in a serum-free medium for mesenchymal stem cells containing 2% FBS (Fukoku Co., Ltd., product number: FKCM301T) at 1 × 10 5 The cells were adjusted to a concentration of 120 cells / mL and seeded at 120 μL / well in a 96-well plate. When the cells reached 100% confluence, the medium was replaced with either 10% FBS (Cytiva, product number SH30910) containing each of the three concentrations shown in Table 4 (No. 1 to 3) or with saline containing 90% FBS (No. 4) as a control (10 samples in total), and the cells were cultured for 7 days (the medium was replaced on the 4th day of culture).

[0051] The substances Nos. 1 to 3 in Table 4 were diluted in three 10-fold dilution series, with an n of 3. After the 7-day culture, SF reagent (Nacalai tesque, product number: 07553-44) was added, and the wavelength was measured using a Nivo multimode plate reader (Revvity) to evaluate cell viability. The reagent was then removed, and calcium deposits were stained with Alizarin Red using the same method as in Example 1. Subsequently, the cells were extracted with 5% formic acid, and the OD at 450 nm / 630 nm was measured using a Nivo multimode plate reader to evaluate calcification using the same method as in Example 10.

[0052] The results are shown in Table 5. As mentioned above, the OD of the formic acid extract was used as an index of calcification, and the OD of the SF reagent was used as an index of cell viability, and the calcification / SF results were compared. Based on the "control" calcification / SF value for control No. 4 (physiological saline containing 90% FBS), the "evaluation value (%)" of each substance No. 1 to 3 was calculated as ("substance" calcification / SF) / ("control" calcification / SF) × 100. Compared to the evaluation value of the control (100%), substances No. 1 to 3 had evaluation values lower than 100%. This demonstrates that this method can measure the calcification inhibition of substances No. 1 to 3. Thus, it is possible to screen for calcification inhibitors using the calcification of cells cultured in 100% serum.

[0053] [Table 5]

[0054] (Example 12) Screening of calcification inhibitors 2 To examine whether calcification inhibitors can be screened using calcification by cells treated with 100% serum at low temperature, 4.2 × 10 ASCs were cultured to 80-90% confluence in the same manner as in Example 8. 5The cells were suspended in 100% FBS at 200 cells / mL, and 360 μL of the cell suspension and 40 μL of each of the substances No. 1 to 3 in Table 4 were added to a 2 mL tube. The tube was then placed in a refrigerator (4 to 10°C) for 4 days for low-temperature treatment. The cells were then seeded into a 96-well plate at 130 μL / well and cultured for 7 days (the medium was changed on the 4th day of culture). After the above 7-day culture, the evaluation values (%) for the substances Nos. 1 to 3 were calculated in the same manner as in Example 11. The results are shown in Table 6.

[0055] [Table 6]

[0056] Compared to the evaluation value of the control (100%), the evaluation values of substances No. 1 to 3 were lower than 100%, which shows that the calcification inhibition by substances No. 1 to 3 can be measured using this method. This suggests that it is possible to screen for calcification inhibitors using calcification induced by cells treated with 100% serum at low temperatures.

[0057] These results demonstrate that low-temperature treatment / cell culture using 85 to 100% serum can induce calcification in specific cells without the use of a differentiation-inducing medium or the like. Furthermore, according to the calcification method of the present invention, calcification can be induced from cells with extremely high efficiency using components derived from autologous cells, without using a differentiation-inducing medium, etc. Furthermore, candidate drugs for calcification inhibitors and calcification promoters can be screened with extremely high efficiency using the calcification.

Claims

1. Mesenchymal stem cells are suspended in 85-100% serum. The suspended mesenchymal stem cells are allowed to stand at 4 to 10°C for at least 4 days. After the incubation, the mesenchymal stem cells are seeded into a culture vessel. A method for calcifying cells by culturing them in a serum-free basal medium, comprising: A method for calcifying cells, wherein the 85 to 100% serum has a calcium concentration of 100 mg / L or more.

2. A method for calcifying cells, comprising culturing the cells in 85 to 100% serum for at least 4 days, thereby calcifying the cells, A method for calcifying cells, wherein the 85 to 100% serum has a calcium concentration of 100 mg / L or more.

3. the serum is of bovine origin; The cells Mesenchymal stem cells, dental pulp cells, periodontal ligament cells, selected from the group consisting of vascular endothelial cells, epidermal keratinocytes, corneal cells, chondrocytes, hair papilla cells, cell lines, and cancer cells; the cell line is HEC293, L929, Balb / 3T3, MC3T3-E1, or Vero cell; The method for calcifying cells according to claim 2, wherein the cancer cells are A549, THP-1, BRL-2H3, or P3U1.

4. the serum is of human origin; The method for calcifying cells according to claim 2 , wherein the cells are mesenchymal stem cells or epidermal keratinocytes.

5. A screening method, comprising calcifying cells by the method for calcifying cells according to any one of claims 1 to 4, and screening for calcification inhibitors using the calcified cells.

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