JP2007175008A - Method for culturing stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for keeping a stem cell collected from a donor for a long period while retaining the multipotency and the differentiation potency of the cell without expressing them and a method for proliferating the cell without impairing the multipotency and the differentiation potency. <P>SOLUTION: The method for culturing the stem cell comprises culturing the stem cell in a liquid medium having specific gravity not precipitating the cell, not adhering the cell to the bottom of a culture vessel and not surfacing the cell and then keeping undifferentiation while retaining the multipotency. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for culturing a stem cell that can be maintained for a predetermined time without impairing the pluripotency and differentiation ability of the stem cell, and in particular, the stem cell can be maintained in an undifferentiated state for a long time without impairing the pluripotency and differentiation ability. Furthermore, the present invention relates to a culture method that proliferates and stem cells that are maintained or proliferated by this culture method.

In recent years, regenerative medicine using stem cells that can differentiate into various cells has attracted attention. For example, in the treatment of blood diseases such as leukemia, aplastic anemia, and congenital immunodeficiency, bone marrow transplantation occupies an important position along with chemotherapy.
Hematopoietic stem cells that play an important role in bone marrow transplantation are the source of blood cells that supply the whole blood system (white blood cells, red blood cells, platelets). A single hematopoietic stem cell can grow in a whole blood cell line while undergoing self-renewal and differentiation.

Since regenerative medicine uses the differentiation ability of undifferentiated stem cells, a large amount of stem cells is required for treatment, but there are very few stem cells contained in various tissues. For example, in bone marrow transplantation performed for treatment of leukemia and the like, hematopoietic stem cells play an important role, but the number is 1/25000 of total bone marrow cells. Therefore, in bone marrow transplantation, a large amount of bone marrow (for example, 600 to 900 mL if the patient's weight is 60 kg) must be collected from the donor, which puts a great burden on the donor and increases the number of registered donors. Has become one of the causes.
In addition, stem cells are difficult to survive for a long period of time in conventional cultures and are differentiated, so cell transplantation is a struggle with time and has become a dilemma in modern medicine.

For these reasons, the possibility of long-term maintenance culture or amplification culture of hematopoietic stem cells has been sought for research development and clinical application of hematopoietic stem cells (see, for example, Non-Patent Document 1 or 2). At present, no technology has been developed to safely maintain and amplify genes without introducing them.
Ueda T, Tsuji K, Yoshino H, Ebihara Y, Yagasaki H, Hisakawa H, Mitsui T, Manabe A, Tanaka R, Kobayashi K, Ito M, Yaskawa K, Nakahata T, “Ueda T, Tsuji K, Yoshino H, Ebihara Y, Yagasaki H, Hisakawa H, Mitsui T, Manabe A, Tanaka R, Kobayashi K, Ito M, Yasukawa K, Nakahata T. ”, Expansion of Human NOD / SCID-Lipoprating Cells Stem cell factor, Flk2 / Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor “Expansion of human NOD / SCID-repopulating cells by stem cell factor, Flk2 / Flt3 ligand, thrombopoietin, IL-6 , and soluble IL-6 receptor. ”, Journal Clinical Investigation“ Journal Clinical Investigation ”, 2000, 105, 1013-1021 Patrick Stiff, Bohao Chen, Wilbur Franklin, David Oldenberg, Eric Hassy, Robert Bayer, Elizabeth Chapal, Judy Deauville, Rankemel Mandaram, Deepak Malhotra, Thomas Mueller, Earl Douglas Armstrong, And Alan Smith “Patrick Stiff, Bohao Chen, Wilbur Franklin, David Oldenberg, Eric Hsi, Robert Bayer, Elizabeth Shpall, Judy Douville, Ramkumar Mandalam, Deepak Malhotra, Thomas Muller, R. Douglas Armstrong, and Alan Smith ”, Autologous Transplantation of Exo Vivo Expanded Bone Mallow Sell's Groun From Small Small Quartz After High-Dose Chemotherapy For Breast Cancer Autologous transplantation of ex vivo expanded bone marrow cells grown from small aliquots after high-dose chemotherapy for breast cancer. ", Brad" Blood ", 2000 years, 95 Nos., Pp. 2169-2174

  An object of the present invention is to solve the above-mentioned problems, and a stem cell culturing method for maintaining a stem cell collected from a donor for a long period of time without expressing it while maintaining its pluripotency and differentiation potential Furthermore, another object of the present invention is to provide a method for culturing stem cells that can be grown without impairing their pluripotency and differentiation ability.

As a result of intensive studies in order to solve the above problems, the present inventors have not completely settled and floated stem cells such as hematopoietic stem cells taken out from a living body by setting the culture medium to a specific specific gravity. It has been found that, by creating a culture environment similar to the condition, that is, the in vivo environment, the stem cells can be maintained or proliferated for a long period of time without being differentiated. The present invention has been made based on this finding.
That is, the present invention
(1) It is characterized in that stem cells are cultured in a liquid medium having a specific gravity in which they do not settle, do not adhere to the bottom of the culture vessel, and do not float, and maintain undifferentiation while maintaining pluripotency. A method for culturing stem cells,
(2) The stem cell culture method according to (1), wherein the specific gravity of the liquid medium is 1.01 to 1.08 g / mL,
(3) The stem cell culture method according to (1) or (2), wherein the stem cell is a hematopoietic stem cell, a mesenchymal stem cell, a neural stem cell, a hepatic stem cell, a vascular stem cell, or an embryonic stem cell,
(4) The specific gravity of the liquid medium is adjusted by containing colloidal silica coated with polyvinylpyrrolidone or a water-soluble copolymer of sucrose and epichlorohydrin in the liquid medium. The method for culturing a stem cell according to any one of (1) to (3),
(5) Stem cells obtained by the culture method according to any one of (1) to (4), maintaining pluripotency, and (6) colloidal silica coated with polyvinylpyrrolidone, or sucrose; The present invention provides a stem cell culture liquid medium in which the specific gravity of the liquid medium is 1.01 to 1.08 g / mL using a water-soluble copolymer with epichlorohydrin.

The stem cell culturing method of the present invention can maintain or proliferate stem cells in an undifferentiated state without requiring a special culturing apparatus or complicated operation.
According to the stem cell culturing method of the present invention, the amount of stem cells collected from a donor necessary for treatment or the like can be reduced, so that the burden on the donor can be reduced, and the stem cell can be transported and transplanted to a remote patient. And the recipient can receive regenerative medicine through safer cell transplantation.
Stem cells obtained by the culture method of the present invention can be used as transplanted donor cells.
The liquid medium for culturing stem cells of the present invention can maintain or proliferate stem cells in an undifferentiated state without requiring complicated operations.

Embodiments of the present invention will be described below.
Stem cells cultured by the culture method of the present invention include hematopoietic stem cells, mesenchymal stem cells, vascular stem cells, neural stem cells, hepatic stem cells, embryonic stem cells (Embryonic stem cells, so-called ES cells) and other types of cells. If it is an undifferentiated cell with the ability to do, it will be applied without limitation.
The hematopoietic stem cells can be applied to bone marrow transplantation that has already been established as a treatment method for leukemia and the like. The stem cells such as the hematopoietic stem cells and mesenchymal stem cells are useful as carrier cells for gene therapy.

  In the present specification and claims, hematopoietic stem cells refer to undifferentiated cells having the ability to differentiate into all types of blood cells and the ability to reconstruct hematopoiesis. Refers to cells that are present in peripheral blood in a small amount. More specifically, if it is a mouse-derived cell, for example, it is a cell expressing an undifferentiated marker such as c-kit or Sca-1 on its cell surface, and if it is a human-derived cell For example, cells expressing undifferentiated markers such as CD34 and CD117 on the cell surface.

In the present invention, the specific gravity of the liquid medium is set to a specific gravity at which stem cells do not settle, do not adhere to the bottom of the culture vessel, and do not float.
In the present invention, “specific gravity in which stem cells do not settle, do not adhere to the bottom of the culture vessel, and do not float” means that the stem cells settle and do not adhere to the bottom of the culture vessel, or stem cells are cells on the bottom. The specific gravity that does not adhere and does not float on the liquid surface of the liquid medium.
In the present invention, the liquid medium whose specific gravity is adjusted can be used without particular limitation as long as it is used for conventional cell culture. For example, Dulbecco's modified Eagle medium (DMEM), the minimum essential Examples thereof include a medium (MEM) and RPMI1640. These usually have a specific gravity of about 1.00 g / mL.
In the present invention, it is necessary to adjust the specific gravity of the liquid medium to a predetermined value.
In the present invention, the specific gravity of the liquid medium is preferably 1.01 g / mL or more, more preferably 1.01-1.08 g / mL, still more preferably 1.01-1.06 g / mL, 1.040-1 0.055 g / mL is particularly preferred.

The method for adjusting the specific gravity of the liquid medium is not particularly limited, but the method for allowing the liquid medium to contain a substance that is inactive with respect to stem cells and can adjust the specific gravity as described above without changing its osmotic pressure. Etc.
Specific examples of the substance capable of adjusting the specific gravity without changing the osmotic pressure of the stem cells described above include colloidal silica coated with polyvinylpyrrolidone such as Percoll (trade name, manufactured by Pharmacia), Ficoll (trade name, manufactured by Pharmacia). ), Water-soluble copolymers of sucrose and epichlorohydrin such as Ficoll Conlay and Ficoll Hipack (both are trade names).
The liquid medium for culturing stem cells of the present invention uses a colloidal silica coated with polyvinyl pyrrolidone or a water-soluble copolymer of sucrose and epichlorohydrin, and the specific gravity of the liquid medium is 1.01-1. It was characterized by being 08 g / mL, 1.01-1.06 g / mL is preferable, and 1.040-1.055 g / mL is more preferable.

In addition to chemical components such as sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, fatty acids, sugars, etc., these liquid media increase serum and cell Biological components such as growth factors (cytokines) can also be contained. Specific examples of cell growth factors include Flt-3-ligand, thrombopoietin (TPO), stem cell factor (SCF), erythropoietin (EPO), and interleukin-6 (IL-6). Is the concentration used in conventional cell culture. However, adding biological components such as serum and cell growth factors may cause infections such as BSE and cancerous cells, so it is better not to add these biological components if possible.
In the present invention, the culture vessel is not particularly limited, and examples thereof include a culture flask, a dish, and a plate. The incubation method is not particularly limited, and examples thereof include shaking culture, stationary culture, and stirring culture.
The incubation temperature may be the same as the normal mammalian cell incubation temperature, preferably 35 to 38 ° C, more preferably 37 ° C.
Stem cells obtained by the culture method of the present invention can be confirmed to be stem cells by confirmation tests using cell surface markers, functional evaluation by transplantation confirmation tests, and the like.

  The stem cells obtained as described above are transplanted into a patient for blood diseases such as bone marrow transplantation and organ regeneration, for example, by infusion. It should be noted that prior to transplantation, histocompatibility must be examined in the same manner as for conventional stem cells.

Next, the mechanism by which stem cells are maintained and proliferated without differentiation for a long period of time by the stem cell culture method of the present invention is considered as follows based on the study of the present inventors.
Cell culture (adherence junction) is suppressed by setting the specific gravity of the liquid medium so that the stem cells do not settle and do not float, and β-catenin acting on the interaction between the adhesion molecule cadherin and the cytoskeleton is inside the stem cells. (See (1-4) in Example 1). The accumulated β-catenin is thought to be involved in stem cell self-renewal.
The adherence junction is ubiquitously present in vertebrate cells, and in particular, cell layer separation, compartmentalization, cell grouping of various stem cells (eg, embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells) during the ontogeny stage It plays an important role in so-called sorting out such as binding (for example, Settleman J. Tension precedes commitment-even for a stem cell. Mol Cell. 2004 Apr 23; 14 (2): 148-50 Edited by Ichiro Yahara, Toshinori Ide and Joichiro Tsukida et al., “Cell Engineering” (Maruzen Co., Ltd., published on March 20, 1991), pages 205-222.)

EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.
Example 1
Mouse bone marrow cells collected as described below were cultured for 7 days.
After disinfecting the mouse (C57BL / 6, 6 weeks old or older, manufactured by CLEA Japan) with cervical dislocation and immersing the whole body in 70% alcohol solution, the femur and tibia are aseptically removed, and the muscle tissue around the bone is removed. After removing as much as possible, both ends of the epiphysis were excised and placed in a petri dish filled with phosphate buffer. The bone marrow tissue was washed away with a syringe with a needle to which the phosphate buffer had been aspirated to recover the bone marrow cell suspension. The collected bone marrow cell suspension was centrifuged (1,500 rpm, 5 minutes) to collect bone marrow cells.

For culturing the bone marrow cells, Dulbecco's modified Eagle medium (DMEM) was used as the basic culture solution, and 20 ng / ml human flt-3-ligand and 20 ng / ml human thrombopoietin were added. 1 × 10 ⁶ bone marrow cells were cultured at 37 ° C. in 1 mL of the culture solution in the culture vessel 24-well plate. As a high specific gravity culture solution, Percoll (trade name, manufactured by Pharmacia) was added to the basic culture solution, and examination was performed under six conditions in the range of specific gravity of 1.004 g / mL to 1.065 g / mL.
Differentiation markers (Lineage markers: CD4, CD8, B220, Mac1, Gr1, TER119) negative, Sca-1 antigen positive and c-Kit positive cells (number of cells and hematopoietic stem cell fraction after 3 and 7 days of culture) Hereinafter, LSK cells were simply measured by a flow cytometer (trade name FACSCalibur, manufactured by Becton Dickinson) by the following antibody staining method, and colony-forming ability and bone marrow transplantation experiments on mice were performed as functional evaluations.

<Antibody staining method>
Each culture solution is collected, cells are separated by a centrifuge, resuspended in a phosphate buffer, and then the biotinylated Lineage antibody (CD4, CD8, B220, Mac1, Gr1, TER119) is added to the cell suspension. And then stained with streptavidin-PerCP (peridinin chlorofin complex protein).
Further, an anti-c-kit antibody (Pharmingen) labeled with allophycocyanin (APC) and an anti-Sca-1 antibody (Pharmingen) labeled with phycoerythrin (PE) are added to a phosphate buffer to form an antibody. A staining solution was prepared.
The number of cells stained with antibodies in this way was counted using the flow cytometer. The flow cytometer was used in accordance with the attached manual.

<Result>
(1) Change in the number of cells (1-1) Change in the total number of bone marrow cells FIG. 1A is a graph showing the change in the number of total bone marrow cells in each specific gravity culture solution, counted using a Birkelturk calculator. is there.
As indicated by the bar graph on day 0 in FIG. 1a, most of the 1 × 10 ⁶ bone marrow cells at the start of the experiment are already differentiated. Therefore, the number of cells decreases when cultured as shown by the bar graph having a specific gravity of 1.004 g / mL on days 3 and 7 in FIG.
However, as shown in the bar graph of specific gravity 1.053 g / mL on days 3 and 7 in FIG. 1a, when the culture medium was cultured at a specific gravity of 1.053 g / mL, the cell number decrease after 7 days was relatively small.
(1-2) Transition of hematopoietic stem cell surface markers possessed by LSK cells FIG. 1 b is a graph showing the transition of the number of hematopoietic stem cell surface markers in each specific gravity culture solution, counted using the flow cytometer.
As apparent from FIG. 1b, when the hematopoietic stem cell surface marker possessed by the LSK cells in the cultured cells was evaluated, it was found that the LSK cells were amplified after 7 days under the condition that the culture medium specific gravity was 1.053 g / mL. I understand.

(1-3) Transition of the number of colony-forming cells Count the number of bone marrow cells (cultured on the 0th and 7th) suspended in phosphate buffer, resulting in 10 ⁵ to 10 ⁶ cells / mL per original cell As described above, the solution was diluted with a phosphate buffer at various concentrations. Then, 1/10 volume of this cell suspension was added to a methylcellulose medium for colony assay (trade name METHOCULT GFM3434, manufactured by STEMCELL TECHNOLOGIS), mixed well by shaking (10 ⁴ to 10 ⁵ cells / mL), 96 100 μL each was dispensed into a well culture dish (10 ³ to 10 ⁴ cells / well). The dispensed bone marrow cells were cultured for 1 week, the number of colonies formed in each well was counted, and the number of colony forming cells per 1000 bone marrow cells was calculated.
FIG. 1c is a diagram showing the number of colony forming cells per 1000 bone marrow cells in each density medium.
As apparent from FIG. 1c, the number of colony forming cells based on LSK cells was amplified after 3 days under the condition that the colony forming ability as a functional evaluation was 1.053 g / mL.

(1-4) Intracellular increase of β-catenin FIG. 2 is an electrophoretic diagram showing the band of β-catenin in stem cells in each specific gravity culture solution after 7 days of culture.
As is clear from FIG. 2, in the lane having a culture medium specific gravity of 1.053 g / mL, the β-catenin band near 90 kDa appears most intensely, indicating that β-catenin is accumulated most in the stem cells.
This indicates that the accumulated β-catenin acts to maintain and amplify the stem cells without differentiation.

(2) Examination with Purified Hematopoietic Stem Cells a-1, b-1, and c-1 in FIG. 3 are immediately after purification, 7 days after culture (specific gravity 1.004), and 7 days after culture (specific gravity 1.053), respectively. , Histogram of counts against differentiation marker negative intensity.
3, a-2, b-2, and c-2 were developed from a-3, b-3, and c-3, respectively, immediately after purification, after 7 days of culture (specific gravity 1.004), and after 7 days of culture ( It is a two-dimensional plot showing the expression pattern of c-kit and Sca-1 measured using the flow cytometer at a specific gravity of 1.053).
Recognition gates a-3 and a-4, b-3 and b-4, c-3 and c-4 correspond to LSK cells, respectively.

Using the flow cytometer, LSK cells having hematopoietic stem cell surface markers were purified. A purity of 95% or more was used. Specifically, the Lineage marker negative cells shown in the recognition gate a-3 were collected, and 100 purified hematopoietic stem cells were obtained.
As is clear from b-1 and b-2 in FIG. 3, when cultured at a culture medium specific gravity of 1.004 g / mL, almost no cells remained after 7 days.
On the other hand, as apparent from c-1 and c-2 in FIG. 3, when the specific gravity of the culture solution is 1.053 g / mL, the number of cells becomes 600 to 1250 after 7 days, that is, about 10 times, most of them. LSK cells.

(3) Hematopoietic stem cell transplantation test In this transplantation test, hematopoietic stem cells of GFP (green fluorescent protein) transgenic mice were used as donor cells.
In order to confirm that the differentiation ability of the proliferated hematopoietic stem cells after 7 days of culture was not impaired, the cells after 7 days of culture were transplanted as donor cells into mice whose hematopoietic function was reduced by irradiation (3 Gy).
Mouse peripheral blood 12 weeks after transplantation was collected, and the presence or absence of engrafted cells was confirmed with the flow cytometer.

4a is a diagram showing the results of transplantation of bone marrow cells before culture, and FIG. 4b is a diagram showing the results of transplantation of cells after 7 days of culture at a culture medium specific gravity of 1.004 g / mL. FIG. 4c is a diagram showing the results of transplanting cells after 7 days of culture at a culture medium specific gravity of 1.053 g / mL. In c of FIG. 4, 10 ³ or more counts of the horizontal axis indicates the count of GFP from engraftment cells.
As is apparent from FIGS. 4a and 4b, the cells before culturing and the cells cultured at a culture medium specific gravity of 1.004 g / mL hardly engrafted.
On the other hand, as apparent from the detection result of the GFP-positive cells in FIG. 4c, the cells cultured at a culture medium specific gravity of 1.053 g / mL were engrafted, so that hematopoietic stem cells were amplified under these conditions. I was able to confirm.
From the results of Example 1, the specific gravity of the culture solution is set to a specific range, and a condition in which hematopoietic stem cells do not settle or float completely, that is, a culture environment similar to the biological environment, is created in bone marrow cells. It can be seen that few hematopoietic stem cells can be cultured while maintaining their differentiation ability for a long time, and the target stem cells can be selectively amplified.

(Example 2)
Human mesenchymal stem cells (hereinafter simply referred to as hMSCs) (trade name PT-2501, manufactured by Cambrex Bio Sciences Walkersville, Inc.) were cultured for 7 days. The basic culture medium was minimal essential medium (MEM) and 10% fetal bovine serum (FBS) was added. 1 × 10 ⁵ cells were cultured in 1 mL of the culture solution. As a high specific gravity culture solution, percoll was contained in the basic culture solution and examined under five conditions in the specific gravity range of 1.004 g / mL to 1.053 g / mL.
Seven days after culturing, the number of cells and the fraction of hMSCs, CD14, CD34, CD45 negative, CD29 positive and CD44 positive cells (that is, cells having hMSCs surface markers) were measured with the flow cytometer.
FIG. 5a is a graph showing the total number of cells in each specific gravity culture solution after 7 days of culture. FIG. 5b is a graph showing the number of cells having the hMSCs surface marker using the flow cytometer.
The number of cells at 1 × 10 ⁵ cells at the start of the experiment decreases when cultured.
However, as apparent from FIG. 5a, when the culture medium was cultured at a specific gravity of 1.017 g / mL, the decrease in the number of cells after 7 days was suppressed.
As is apparent from FIG. 5b, when the hMSCs surface marker in cultured cells was evaluated using the flow cytometer, the number of hMSCs after 7 days was higher than that of the other when the culture medium specific gravity was 1.017 g / mL. It was a lot.
Even if the commercially available hMSCs used were cultured using a normal growth medium, the cells decreased after 7 days.
However, as shown in Example 2, when the specific gravity of the culture solution was within a specific range, the decrease was suppressed and the number of hMSCs was large. It can be seen that mammalian mesenchymal stem cells can be maintained and amplified without differentiation for a long period of time as in the case of the mammalian hematopoietic stem cells shown in Example 1 by setting the specific gravity of the culture medium within a specific range.

FIG. 1a is a graph showing the transition of the total number of bone marrow cells in each specific gravity culture solution. FIG. 1b is a graph showing the change in the number of hematopoietic stem cell surface markers in each density medium. FIG. 1c is a diagram showing the number of colony forming cells per 1000 bone marrow cells in each density medium. FIG. 2 is an electrophoretogram showing the stem cell β-catenin band (90 KDa) in each density medium. The a-1, b-1, and c-1 in FIG. 3 respectively correspond to the differentiation (Lineage) marker negative intensity immediately after purification, 7 days after culture (specific gravity 1.004), and 7 days after culture (specific gravity 1.053). It is a histogram of count numbers. The a-2, b-2, and c-2 in FIG. 3 were developed in a-3, b-3, and c-3, respectively, immediately after purification, after 7 days of culture (specific gravity 1.004), and after 7 days of culture ( It is a two-dimensional plot showing the expression pattern of c-kit and Sca-1 measured using the flow cytometer at a specific gravity of 1.053). 4a is a diagram showing the results of transplantation of bone marrow cells before culture, and FIG. 4b is a diagram showing the results of transplantation of cells after 7 days of culture at a culture medium specific gravity of 1.004 g / mL. FIG. 4c is a diagram showing the results of transplanting cells after 7 days of culture at a culture medium specific gravity of 1.053 g / mL. FIG. 5a is a graph showing the total number of cells in each specific gravity medium after 7 days of culture. FIG. 5b is a graph showing the number of cells having the hMSCs surface marker using the flow cytometer.

Claims (6)

  Stem cells are cultured in a liquid medium with a specific gravity so that they do not settle, do not adhere to the bottom of the culture vessel, and do not float, and maintain undifferentiation while maintaining pluripotency Culture method.   The method for culturing stem cells according to claim 1, wherein the specific gravity of the liquid medium is 1.01-1.08 g / mL.   The stem cell culture method according to claim 1 or 2, wherein the stem cells are hematopoietic stem cells, mesenchymal stem cells, neural stem cells, hepatic stem cells, vascular stem cells, or embryonic stem cells.   The specific gravity of the liquid medium is adjusted by containing colloidal silica coated with polyvinylpyrrolidone or a water-soluble copolymer of sucrose and epichlorohydrin in the liquid medium. The method for culturing a stem cell according to any one of -3.   The stem cell which maintained the pluripotency obtained by the culture method of any one of Claims 1-4. A liquid medium for stem cell culture using a colloidal silica coated with polyvinylpyrrolidone or a water-soluble copolymer of sucrose and epichlorohydrin, and the specific gravity of the liquid medium is 1.01-1.08 g / mL.