Serum-free medium and method for expanding hematopoietic stem cells
阅读说明：本技术 无血清培养基及扩增造血干细胞的方法 (Serum-free medium and method for expanding hematopoietic stem cells ) 是由 罗玮瑜 曾佩娸 余昭勋 于 2018-06-05 设计创作，主要内容包括：本发明提供一种无血清培养基及扩增造血干细胞的方法。所述无血清培养基包括无血清基础培养基、细胞激素、脐带间质干细胞条件培养基以及辅助成分。所述细胞激素包括干细胞因子、血小板生成素以及造血生长因子Flt3配体。所述脐带间质干细胞条件培养基源自于经培养的人类脐带间质干细胞。所述辅助成分包括维生素C、维生素E、或是维生素C及维生素E的组合。(The invention provides a serum-free culture medium and a method for amplifying hematopoietic stem cells. The serum-free culture medium comprises a serum-free basal culture medium, cell hormones, a cord mesenchymal stem cell conditioned medium and auxiliary components. The cytokines include stem cell factor, thrombopoietin, and hematopoietic growth factor Flt3 ligand. The umbilical cord mesenchymal stem cell conditioned medium is derived from cultured human umbilical cord mesenchymal stem cells. The auxiliary component comprises vitamin C, vitamin E, or the combination of vitamin C and vitamin E.)
1. a serum-free medium for expansion of hematopoietic stem cells, comprising:
Serum-free basal medium;
cytokines including stem cell factor, thrombopoietin, and hematopoietic growth factor Fms-related tyrosine kinase 3 ligand;
Umbilical cord mesenchymal stem cell conditioned medium derived from cultured human umbilical cord mesenchymal stem cells; and
An adjunct ingredient comprising vitamin C, vitamin E, or a combination of vitamin C and vitamin E.
2. The serum-free medium according to claim 1, wherein the auxiliary component is vitamin C.
3. The serum-free medium according to claim 1, wherein the auxiliary component is vitamin E.
4. The serum-free medium according to claim 1, wherein the adjunct ingredients comprise a combination of vitamin C and vitamin E.
5. The serum-free medium according to claim 4, wherein the adjunct ingredient further comprises estradiol.
6. The serum-free medium according to claim 1, wherein the umbilical cord mesenchymal stem cell conditioned medium is prepared by a method comprising the steps of:
(a) Culturing human umbilical mesenchymal stem cells in a cell culture medium; and
(b) The cell culture medium was separated by centrifugation, and then the supernatant was collected to obtain a conditioned medium.
7. The serum-free medium according to claim 6, further comprising step (c): concentrating the conditioned medium using a 5 kilodalton to 10 kilodalton blocking membrane to obtain a concentrated umbilical cord mesenchymal stem cell conditioned medium.
8. The serum-free medium according to claim 7, wherein in step (c), the umbilical cord mesenchymal stem cell conditioned medium is concentrated 7-fold to 12-fold by volume.
9. The serum-free medium of claim 8, wherein the umbilical cord mesenchymal stem cell conditioned medium is concentrated to a protein concentration of 50-200 mg/ml.
10. The serum-free medium of claim 1, wherein the molecular weight of the protein component in the umbilical cord mesenchymal stem cell conditioned medium is greater than 5 kilodaltons.
11. the serum-free medium of claim 1, wherein the cytokines further comprise interleukins type 3 and type 6.
12. The serum-free medium of claim 1, wherein the cytokine further comprises a granulocyte colony stimulating factor.
13. The serum-free medium according to claim 1, wherein the umbilical cord mesenchymal stem cell conditioned medium comprises a hematopoietic stem cell expansion-related protein selected from at least one of the following groups: a cysteine-rich acidic secreted protein, follistatin-related protein 1, metalloproteinase inhibitor 1, macrophage colony stimulating factor 1 receptor, periostin, galectin 1, CD166 antigen, distal upstream element binding protein 1, or a combination thereof.
14. A method of expanding hematopoietic stem cells, comprising the steps of:
Providing a serum-free medium prepared by mixing a serum-free basal medium with cytokines, a cord mesenchymal stem cell conditioned medium and auxiliary components, wherein the cytokines comprise stem cell factors, thrombopoietin and hematopoietic growth factor Fms-associated tyrosine kinase 3ligand, the cord mesenchymal stem cell conditioned medium is derived from cultured human cord mesenchymal stem cells, and the auxiliary components comprise vitamin C, vitamin E, or a combination of vitamin C and vitamin E; and
Culturing hematopoietic stem cells in the serum-free medium for a first period of time.
15. The method for expanding hematopoietic stem cells of claim 14, further comprising supplementing the serum-free medium by 50-80% after the first period of time and continuing the culture for a second period of time.
16. The method of expanding hematopoietic stem cells of claim 15, wherein the first period of time and the second period of time are in the range of 1-20 days.
17. The method for expanding hematopoietic stem cells according to claim 14, wherein the umbilical cord mesenchymal stem cell-conditioned medium is prepared by a method comprising the steps of:
(a) Culturing human umbilical mesenchymal stem cells in a cell culture medium; and
(b) The cell culture medium was separated by centrifugation, and then the supernatant was collected to obtain a conditioned medium.
18. The method for expanding hematopoietic stem cells according to claim 17, further comprising the step (c): concentrating the conditioned medium using a 5 kilodalton to 10 kilodalton blocking membrane to obtain a concentrated umbilical cord mesenchymal stem cell conditioned medium.
Technical Field
The invention relates to a serum-free culture medium, in particular to a serum-free culture medium and a method for amplifying hematopoietic stem cells.
Background
Umbilical cord blood transplantation (UBCT) is a new therapeutic approach to treat previously incurable diseases. However, cord blood transplantation in adults is limited by the small number of primitive Hematopoietic Stem Cells (HSCs) available in each transplanted individual. A small number of primitive hematopoietic stem cells may cause delayed engraftment after transplantation. Currently, attempts to expand umbilical cord blood precursor cells ex vivo (ex vivo) have not been very successful. Ex vivo expansion typically results in the expansion of mature hematopoietic stem cells, while immature hematopoietic stem cells are not. In addition, ex vivo expansion of cord blood hematopoietic stem cells may result in defects such as promotion of apoptosis, destruction of bone marrow homing (marrow homing), and initiation of cell cycle (cell cycling).
The difficulty in ex vivo expansion of hematopoietic stem cells lies in the need for various conditioning factors for the growth and proliferation of the original hematopoietic stem cells. Early studies showed that ex vivo growth of hematopoietic stem cells requires cytokines and hematopoietic growth factors produced by other tissues present in serum. Such factors include, for example, erythropoietin, interleukin type 3 (IL-3), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), Stem Cell Factor (SCF), and interleukin type 11 (interleukin-11; IL-11), and the like.
Due to the requirements for various complex factors, it is difficult to generate sufficient numbers of hematopoietic stem cells and to avoid differentiation of the starting cell population. In vitro (in vitro) studies have found that the self-renewal and differentiation of hematopoietic stem cells in cell culture medium is difficult to control. The existing methods using cytokines (cytokines) cannot effectively and reliably help the expansion of immature stem cells in the culture medium, which indicates that other factors (besides cytokines) are required to help the expansion.
Most primitive hematopoietic stem cells usually have the presence of CD34 on their cell membrane. CD34 is a surface glycoprotein of unknown function. Cells bearing the CD34 antigen are thought to be responsible for multi-lineage engraftment (multi-lineageengement). Although CD34 is present on most proliferating cells, CD34 is rarely found on other cells, for example, only about 1% of collected Monocytes (MNC) are found to have CD34 present. Since the proliferative hematopoietic stem cells are cells of CD34+, hematopoietic expansion starting from cells of CD34+ has greater potential. However, starting from hematopoietic expansion of CD34+ cells alone will not succeed due to the lack of helper cells that may provide cytokines and other stimulatory factors. Therefore, expansion of hematopoietic stem cells is usually performed in the presence of serum and other tissues as feeder layers (feeders).
However, the use of serum is undesirable as it can cause possible contamination and adverse immune reactions. Therefore, serum-free alternatives are actively being sought. For example, a serum-free or serum-depleted (serum-depleted) medium for culturing hematopoietic stem cells and bone marrow stromal cells is disclosed in U.S. Pat. No. 5,405,772, and a serum-free medium for expanding CD34+ hematopoietic stem cells and bone marrow lineage (myeloididerige) cells is disclosed in U.S. Pat. No. 6,733,746.
Although these techniques have provided useful media for the expansion of hematopoietic stem cells, there is still a need for better media and methods for expanding hematopoietic stem cells.
Disclosure of Invention
The invention provides a serum-free culture medium which can be used for expanding hematopoietic stem cells.
In one embodiment of the present invention, a serum-free medium for expansion of hematopoietic stem cells is provided. The serum-free medium comprises a serum-free basal medium, a cytokine, an umbilical cord mesenchymal stem cell conditioned medium (umbilical cordisenchyl stem cell conditioned medium) and an auxiliary component. The cytokines include Stem Cell Factor (SCF), Thrombopoietin (TPO), and hematopoietic growth factor (Fms) -related tyrosine kinase 3ligand (Flt 3L). The umbilical cord mesenchymal stem cell conditioned medium is derived from cultured human umbilical cord mesenchymal stem cells. The auxiliary component comprises vitamin C, vitamin E, or the combination of vitamin C and vitamin E.
According to some embodiments of the invention, the serum-free basal medium may be any serum-free basal medium suitable for cell culture. Many suitable media are known in the art. For example, in U.S. Pat. No. 5,405,772, a serum-free or serum-depleted (serum-depleted) medium for culturing hematopoietic stem cells and bone marrow stromal cells is disclosed. Serum-free media for the expansion of CD34+ hematopoietic stem cells and cells of the myeloid lineage (myeloid linkage) are disclosed in US6,733,746. A method for identifying the optimal composition of serum-free eukaryotic cell culture media supplements is disclosed in US8,762,074. The disclosures of these patents are incorporated by reference in their entirety. The basal media disclosed in these prior art references may be used with embodiments of the present invention.
In some embodiments of the invention, the adjunct ingredient is vitamin C.
In some embodiments of the invention, the adjunct ingredient is vitamin E.
In some embodiments of the invention, the adjunct ingredient comprises a combination of vitamin C and vitamin E.
In some embodiments of the invention, the adjunct ingredient further comprises estradiol (E2).
In some embodiments of the invention, the umbilical cord mesenchymal stem cell conditioned medium is prepared by a method comprising the steps of: (a) culturing human umbilical mesenchymal stem cells in a cell culture medium; and (b) isolating the cell culture medium to obtain a conditioned medium.
In some embodiments of the present invention, the method for preparing the umbilical cord mesenchymal stem cell-conditioned medium further comprises the step (c): concentrating the conditioned medium using a 5 kilodalton to 10 kilodalton blocking membrane (cut-off membrane) to obtain a concentrated umbilical cord mesenchymal stem cell conditioned medium.
In some embodiments of the invention, in step (c), the umbilical cord mesenchymal stem cell-conditioned medium is concentrated 7-fold to 12-fold.
In some embodiments of the invention, the umbilical cord mesenchymal stem cell conditioned medium is concentrated to a protein concentration of 100 mg/ml. The umbilical cord mesenchymal stem cell conditioned medium may be concentrated to a desired protein concentration, for example, 50-200mg/ml, preferably 100-150mg/ml (e.g., 100mg/ml, 110mg/ml, 120mg/ml, 130mg/ml, 140mg/ml or 150 mg/ml).
In some embodiments of the invention, the molecular weight of the proteinaceous component in the conditioned medium of umbilical cord mesenchymal stem cells is greater than 5 kilodaltons. This can be achieved, for example, by means of dialysis or ultrafiltration using membranes with a blocking molecular weight of 5 kilodaltons.
In some embodiments of the invention, the cytokines further include interleukins of type 3 (interleukin 3; IL-3) and type 6 (interleukin 6; IL-6).
in some embodiments of the invention, the cytokine further comprises a granulocyte colony stimulating factor (G-CSF).
In some embodiments of the invention, the main composition of the serum-free basal medium comprises human albumin (human albumin), albumin-related proteins and peptides, insulin, salts, polysaccharides, amino acids, vitamins, buffers containing phenol red (phenol-red), L-glutamine (L-glutamine), and β -mercaptoethanol (β -mercaptoethanol).
In some embodiments of the invention, the serum-free basal medium may be a serum-free Stem Cell Growth Medium (SCGM) or X-VIVO 15.
In another embodiment of the present invention, a method of expanding hematopoietic stem cells is disclosed. The method comprises the following steps. Provides a serum-free culture medium. The serum-free culture medium is prepared by mixing a serum-free basal culture medium with cell hormones, a cord mesenchymal stem cell conditioned medium and auxiliary components, wherein the cell hormones comprise stem cell factors, thrombopoietin and hematopoietic growth factor Fms-related tyrosine kinase 3 ligands, the cord mesenchymal stem cell conditioned medium is derived from cultured human cord mesenchymal stem cells, and the auxiliary components comprise vitamin C, vitamin E or a combination of vitamin C and vitamin E. Culturing hematopoietic stem cells in the serum-free medium for a first period of time.
In some embodiments of the present invention, the method for expanding hematopoietic stem cells further comprises supplementing (replenishing) 50-80% serum-free medium after the first period of time and continuing culturing for a second period of time.
In some embodiments of the invention, hematopoietic stem cells are cultured for a first period of time (e.g., 1-20 days), then supplemented with 50-80% serum-free medium and cultured for a second period of time (e.g., 1-20 days). This replenishment and cleaning may be repeated multiple times.
In view of the above, the serum-free medium of the present invention comprises at least a serum-free basal medium, a cytokine, a cord mesenchymal stem cell conditioned medium, and an auxiliary component, and therefore, expansion of hematopoietic stem cells can be improved.
In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.
FIGS. 1A and 1B show the results of hematopoietic stem cell expansion using a specific growth medium in the presence of a feeder layer.
FIGS. 2A and 2B show the results of evaluation of the effect of adding 4 auxiliary components to a growth medium containing a feeder layer on the expansion of hematopoietic stem cells.
FIGS. 3A and 3B show the results of the effect on the expansion of hematopoietic stem cells by using umbilical cord mesenchymal stem cell conditioned medium instead of feeder layer.
FIGS. 4A and 4B show the results of the effect on hematopoietic stem cell expansion by using the concentrated umbilical cord mesenchymal stem cell-conditioned medium instead of the feeder layer.
FIGS. 5A and 5B show the results of evaluating the effect of adding 4 kinds of auxiliary components to the concentrated umbilical cord mesenchymal stem cell-conditioned medium on the expansion of hematopoietic stem cells.
Fig. 6A and 6B show the results of comparing the fold expansion of the colony forming unit and the fold expansion of the accumulated CD34+ cells in hematopoietic stem cell expansion.
Fig. 7A and 7B show the results of evaluating the effect of estradiol (E2), vitamin C, and vitamin E on hematopoietic stem cell expansion in combination.
fig. 8A and 8B show the results of evaluating the effect of vitamin C on the expansion of hematopoietic stem cells.
Fig. 9A and 9B show the results of evaluation of the effect of vitamin E on hematopoietic stem cell expansion.
Fig. 10A and 10B show the results of evaluation of the effect of the combination of vitamin C and vitamin E on the expansion of hematopoietic stem cells.
FIGS. 11A and 11B show the results of the effect on the expansion of hematopoietic stem cells by supplementing the medium.
Fig. 12A and 12B show the results of the effect on the expansion of hematopoietic stem cells by using different combinations of cytokines.
Fig. 13 shows the relative expansion of CD34+ cells relative to total cell expansion.
FIG. 14 shows the percentage of units evaluated for erythrocyte lineage colony formation in hematopoietic stem cell expansion.
Description of reference numerals:
S1, S3-2, S3-3 and QC: group of
PC: positive control group
SCGM: serum-free stem cell growth medium
IMDM: basal cell culture medium
X-Vivo 15: serum-free basal medium
CTK6/CTK × 6: 6 different cytokines
SP: auxiliary ingredients
SP 3: 3 auxiliary components
SP 4: 4 auxiliary components
UCM: umbilical cord mesenchymal stem cell conditioned medium
SF-UCM: serum-free umbilical cord mesenchymal stem cell conditioned medium
c-SF-UCM: concentrated umbilical cord mesenchymal stem cell conditioned medium
E2: estradiol
And Vit.C: vitamin C
Vit.E: vitamin E
TF: transferrin
Detailed Description
Reference will now be made in detail to the present preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings and the description to refer to the same or like parts.
Embodiments of the invention relate to serum-free media and methods of expanding Hematopoietic Stem Cells (HSCs). According to embodiments of the present invention, the medium used for hematopoietic stem cell expansion does not require serum or other ancillary tissues/cells (e.g., feeder layers). Rather, the desired factor is replaced with a particular component.
The examples of the invention are based on specific media and factors. The serum-free medium of the present embodiment includes at least a serum-free basal medium, a cytokine, a cord mesenchymal stem cell conditioned medium, and an auxiliary component. These components will be described in further detail below.
serum-free basal medium
To avoid possible contamination and adverse immune reactions, the basal medium should be serum-free (serum-free) and free of other tissues or cells. A suitable serum-free basal medium for expanding Hematopoietic Stem Cells (HSCs) according to embodiments of the present invention may be based on any suitable commercially available medium. For example, the following commercial media have been tested.
X-VIVOTM15 is a chemically defined serum-free medium suitable for hematopoietic cell culture, available from Longsha (Lonza; Switzerland).
Different SCGMTM(stem cell growth medium; stem cell growth medium) can be adapted to various cell types. For example, human bone marrow stem cell growth medium is available from Sigma-Aldrich. In thatIn the experimental examples, serum-free SCGM was obtained from CellGenix (accession number 20802-.
Iscove's Modified Dulbecco's Media (IMDM) is a highly concentrated synthetic medium well suited for high density cell culture for rapid proliferation. IMDM is available from many commercial sources, for example from ThermoFisher Scientific. However, IMDM is a synthetic basal cell culture medium that typically requires the addition of serum and other growth hormones for cell growth. In the experimental examples, IMDM can be used as a positive control to compare with the serum-free basal medium described above for assessing cell expansion.
According to an embodiment of the present invention, the medium for hematopoietic stem cell expansion may, for example, include a commercially available special serum-free base medium (SFM), such as SCGM described above. Based on this serum-free basal medium (e.g., SCGM), selected chemicals and cytokines can be added as well as conditioned medium from umbilical cord mesenchymal stem cells (UC-MSC) for evaluation.
Cytokines
In cell expansion experiments, 6 different cytokines (called "cytokines x 6" or "CTK x 6") can be used. The 6 cytokines include recombinant human stem cell factor (rh SCF), recombinant human thrombopoietin (rh TPO), recombinant human hematopoietic growth factor (Fms) related tyrosine kinase 3ligand (recombinant human hematopoietic growth factor Fms-related tyrosine kinase 3 ligand; Flt3L), recombinant human interleukin 3, recombinant human interleukin 6 (rh IL-3), recombinant human interleukin 6 (rh IL-6), and recombinant human granulocyte colony stimulating factor (rh G-CSF).
In one embodiment of the present invention, the concentration of the recombinant human stem cell factor is in the range of 20-300ng/ml, preferably 20-100ng/ml, and more preferably 20-50 ng/ml. The concentration of the recombinant human thrombopoietin is in the range of 10-100ng/ml, preferably 20-100ng/ml, more preferably 20-50 ng/ml. The concentration of the recombinant human hematopoietic growth factor Fms-related tyrosine kinase 3ligand is in the range of 50-300ng/ml, preferably 50-100ng/ml, more preferably 50-80 ng/ml. The concentration of the recombinant human type 3 interleukin is in the range of 1-20ng/ml, preferably 5-15ng/ml, and more preferably 10-15 ng/ml. The concentration of the recombinant human type 6 interleukin is in the range of 10-100ng/ml, preferably 10-50ng/ml, and more preferably 10-30 ng/ml. The concentration of the recombinant human granulocyte colony stimulating factor is in the range of 1-100ng/ml, preferably 1-50ng/ml, and more preferably 1-20 ng/ml.
In a preferred embodiment, the concentration of the recombinant human stem cell factor is 20 ng/ml. The concentration of the recombinant human thrombopoietin is 20 ng/ml. The concentration of the recombinant human hematopoietic growth factor Fms-related tyrosine kinase 3ligand is 50 ng/ml. The concentration of the recombinant human type 3 interleukin is 10 ng/ml. The concentration of the recombinant human type 6 interleukin is 10 ng/ml. The concentration of the recombinant human granulocyte colony stimulating factor is 1 ng/ml.
Umbilical cord mesenchymal stem cell conditioned medium
According to some embodiments of the invention, the umbilical cord mesenchymal stem cell conditioned medium is derived from cultured human umbilical cord mesenchymal stem cells. In one embodiment of the present invention, the umbilical cord mesenchymal stem cell-conditioned medium is prepared by a method comprising the steps of: (a) culturing human umbilical cord mesenchymal stem cells in a serum-free cell culture medium (e.g., serum-free SCGM) for 3-5 days; and (b) separating the serum-free cell culture medium to obtain a serum-free umbilical cord mesenchymal stem cell conditioned medium (hereinafter referred to as "SF-UCM").
The obtained SF-UCM may be further concentrated by the following step (c): concentrating the conditioned medium (SF-UCM) using a 5 kilodalton to 10 kilodalton blocking membrane to obtain a concentrated umbilical cord mesenchymal stem cell conditioned medium (hereinafter referred to as "con. SF-UCM" or "c-SF-UCM").
In one embodiment of the present invention, the step (b) comprises centrifuging the umbilical cord mesenchymal stem cells and the cell culture medium at 500g and 16 ℃ for 10 minutes, then collecting the supernatant to obtain a conditioned medium, and discarding the precipitate. In some other embodiments, step (c) above is for obtaining concentrated umbilical cord mesenchymal stem cell conditioned medium. For example, in step (c), the conditioned medium (SF-UCM) obtained in step (b) is concentrated by using a blocking membrane of 5 kilodaltons to 10 kilodaltons, preferably 5 kilodaltons, to obtain a 7-12 fold (by volume) concentrated conditioned medium. In some embodiments, the umbilical cord mesenchymal stem cell conditioned medium is preferably concentrated 10-fold in step (c) (by volume). The concentrated conditioned medium is filtered using a 0.22 μm filter, and the filtrate is collected to obtain the desired concentrated umbilical cord mesenchymal stem cell conditioned medium (c-SF-UCM). In some embodiments, the umbilical cord mesenchymal stem cell conditioned medium is concentrated to a protein concentration of 50-200mg/ml, preferably to 100-150 mg/ml. In a preferred embodiment, the protein concentration of c-SF-UCM is 100 mg/ml. In some other embodiments, the umbilical cord mesenchymal stem cell conditioned medium is concentrated to obtain a conditioned medium comprising a protein fraction with a molecular weight greater than 5 kilodaltons.
In an exemplary embodiment, identification of the protein components included in the umbilical cord mesenchymal stem cell conditioned medium by proteome analysis (proteomic analysis) may, for example, include hematopoietic stem cell proliferation-related proteins, hematopoietic stem cell homing-related proteins, immunoregulation-related proteins, neuronal development-related proteins, metabolic process-related proteins, cell component-related proteins, vesicle trafficking proteins, SCGM media components, and some other unannotated components. For example, the main 91 proteins identified are detailed in table 1 below, where the table does not list 48 SCGM medium components and 35 unannotated components.
Table 1: list of proteins identified by proteosome analysis in concentrated umbilical cord mesenchymal stem cell conditioned media
In some embodiments, the umbilical cord mesenchymal stem cell conditioned medium comprises a hematopoietic stem cell expansion-related protein selected from at least one of the following groups: cysteine-rich acidic Secreted Protein (SPARC), follistatin-related protein 1, metalloproteinase inhibitor 1, macrophage colony stimulating factor 1 receptor, periostin, galectin 1, CD166 antigen, distal upstream element binding protein 1, or a combination thereof.
Auxiliary ingredients
In some embodiments herein, a variety of supplemental ingredients can be used in serum-free media, including vitamin C, vitamin E, estradiol (E2), and Transferrin (TF). In previous studies, vitamin C and vitamin E have been provided as medical nutritional therapies to adult human hematopoietic stem cell transplant patients to minimize toxicity induced by pretreatment conditions (nutr.
herein, the term "vitamin C" (vit.c) refers to synthetic or natural L-ascorbic acid (L-ascorbyl acid), a bioavailable form thereof or a derivative thereof. In one embodiment, the concentration of vitamin C is in the range of 50-375 μ M, preferably 100-300 μ M, and more preferably 200-300 μ M. In a preferred embodiment, the concentration of vitamin C is 250. mu.M.
In this context, the term "vitamin E" (vit. E) refers to all tocopherols (tocophenols), synthetic or natural (i.e. all steric forms of alpha-, beta-and gamma-tocopherol), their bioavailable forms or derivatives thereof. In one embodiment, alpha-tocopherol is preferred for the purposes of the present invention. In one embodiment, the concentration of vitamin E is in the range of 2-20. mu.M, preferably 2-15. mu.M, more preferably 2-10. mu.M. In a preferred embodiment, the concentration of vitamin E is 2 μ M.
In one embodiment, the concentration of estradiol (E2) is at 10-9-10-8And M is in the range. In a preferred embodiment, the concentration of estradiol is 10-9M。
In one embodiment, the transferrin concentration is in the range of 10-100 μ g/ml, preferably 10-80 μ g/ml, more preferably 10-50 μ g/ml. In a preferred embodiment, transferrin is present at a concentration of 30 μ g/ml.
Examples of the experiments
The following experimental examples will be used to evaluate various factors that may affect hematopoietic stem cell expansion.
experimental example 1: evaluation of hematopoietic Stem cell Medium
To evaluate various media for the in vitro expansion of Hematopoietic Stem Cells (HSCs), IMDM containing essential cytokines, 5% cord serum (cord serum; CS), and a feeder layer were used as controls. Both media (SCGM and X-VIVO15) were tested in the absence of serum (but in the presence of the same cytokines and feeder layers) to see if they could be used as serum-free media. The detailed experimental procedure is as follows.
On day-1, umbilical cord mesenchymal stem cells (UC-MSCs) were seeded in a T-12.5 vessel and cultured as feeder cells in complete culture medium (containing 10% human umbilical cord serum and DMEM). On day 0, CD34+ hematopoietic stem cells were thawed and cultured with the UC-MSC feeder cells at a cell density of 2.5X 104Co-culture at cell/mlCulturing for 12 days, and using the following different culture media: (1) positive control group (PC): contains 5% of umbilical cord serum, 6 kinds of cytokines and hydrocortisone (10)-6M) an IMDM; (2) SCGM group: contains 6 kinds of cell hormones and hydrocortisone (10)-6M) serum-free stem cell growth medium SCGM; and, (3) group X-VIVO 15: contains 6 kinds of cell hormones and hydrocortisone (10)-6M) serum-free basal medium X-VIVO 15. During the 12 day culture period, 50% of the medium and freshly prepared UC-MSC feeder cells were supplemented every 4 days, and the cells (including CD34+ cells) were maintained at 2.5 to 5X 104Cell density of cells/ml. The 6 cytokines used above include recombinant human stem cell factor (rh SCF; 20ng/ml), recombinant human thrombopoietin (rh TPO; 20ng/ml), recombinant human hematopoietic growth factor Fms-related tyrosine kinase 3ligand (rh Flt 3L; 50ng/ml), recombinant human interleukin type 3 (rh IL-3; 10ng/ml), recombinant human interleukin type 6 (rh IL-6; 10ng/ml) and recombinant human granulocyte colony stimulating factor (rh G-CSF; 1 ng/ml). On day 12, the cumulative fold amplification of total cells and the fold amplification of CD34+ (ishag) were calculated, and the experimental results are shown in fig. 1A and 1B. Cells of CD34+ were quantified according to the guidelines of the International Society of blood treatment and transplantation Engineering (ISHAGE) (Sutherland et al, J.Hematother, 1996, Jun:5(3): 213-26).
As shown in fig. 1A and 1B, SCGM was able to better support expansion of hematopoietic stem cells in the absence of serum in the tested media compared to control IMDM, while X-VIVO15 was less effective. In addition, SCGM is a serum-free medium with good efficacy for the expansion of total cells, and especially for the expansion of CD34+ cells, in the presence of a feeder layer. It is currently known that most of the proliferative hematopoietic stem cells are CD34+ cells. Therefore, maintaining the expansion capacity of CD34+ cells is more important than maintaining the growth of total cells. Based on this, SCGM was selected as the basal medium for the serum-free medium of the present invention.
As described above, various cytokines and factors contributed by other cells or tissues are required for the in vitro growth of hematopoietic stem cells. One hypothesis is that true hematopoietic stem cells are essentially fixed tissue cells that exist with other supporting tissues/cells, and that the microenvironment provided by these supporting tissues/cells allows the hematopoietic stem cells to self-renew without differentiation and maturation. In this regard, stromal cells (stromal cells) can provide a wide range of environmental signals conducted by cytokines, extracellular matrix proteins and adhesion molecules, which can be used to control the proliferation, survival and differentiation of hematopoietic progenitor cells (hematopoietic progenitors) and stem cells. Thus, a feeder layer containing stromal cells may be used to provide any of these factors in the in vitro growth of hematopoietic stem cells.
However, the use of tissue or cells as a feeder layer is undesirable as it may introduce contamination or cause adverse immune reactions. The inventors of the present invention have found through experiments that conditioned medium derived from umbilical cord mesenchymal stem cells can replace a feeder layer to support the in vitro expansion of hematopoietic stem cells. The experiments that support these discussions will be explained in detail in the examples that follow.
Experimental example 2: evaluation of Effect of auxiliary ingredients
This example evaluated the effect of adding 4 auxiliary ingredients to the medium containing the feeder layer. The specific experimental procedure is as follows.
On day-1, umbilical cord mesenchymal stem cells (UC-MSCs) were seeded in a T-12.5 vessel and cultured as feeder cells in complete culture medium (containing 10% human umbilical cord serum and DMEM). On day 0, CD34+ hematopoietic stem cells were thawed and cultured with the UC-MSC feeder cells at a cell density of 2.5X 104Cells were co-cultured at/ml for 12 days, with the following different media: (1) positive control group (PC): IMDM containing 5% umbilical cord serum, 6 cytokines and hydrocortisone; (2) SCGM group: SCGM containing 6 cytokines and hydrocortisone; and, (3) SCGM + SP4 group: SCGM containing 6 cytokines, hydrocortisone and 4 auxiliary components. The 6 cytokines and hydrogenated prednisone used in this example were the same as those used in example 1. The 4 auxiliary components (also called SP4 or auxiliary for short)Auxiliary components 4) including vitamin C (250 μ M), vitamin E (2 μ M), and estradiol (10 μ M)-9M) and transferrin (30. mu.g/ml). During the 12 day culture period, 50% of the medium and freshly prepared UC-MSC feeder cells were supplemented every 4 days, and cells (including CD34+ cells) were maintained at 2.5-5X 104Cell density of cells/ml. On day 12, the cumulative fold amplification of total cells and the fold amplification of CD34+ (ishag) were calculated, and the experimental results are shown in fig. 2A and 2B.
As shown in fig. 2A and 2B, these 4 accessory components did not have any significant effect on total cell expansion and expansion of CD34+ cells in the presence of the feeder layer. The possible reason is that some factors secreted by the feeder layer have similar effects to the 4 accessory ingredients in the presence of the feeder layer. Therefore, the addition of 4 additional accessory ingredients in the presence of these factors did not further enhance cell expansion.
Experimental example 3: replacement of feeder layer by serum-free umbilical cord mesenchymal stem cell conditioned medium
To test potential alternatives to feeder layers, we tested the effect of serum-free umbilical mesenchymal stem cell conditioned medium (SF-UCM) derived from umbilical mesenchymal stem cells (UC-MSC). The serum-free umbilical cord mesenchymal stem cell conditioned medium is prepared, for example, by the above-mentioned method. The method comprises the following steps: (a) culturing umbilical cord mesenchymal stem cells in serum-free cell culture medium (e.g., serum-free SCGM); and (b) isolating the conditioned medium. The experimental results of replacing the feeder layer with umbilical cord mesenchymal stem cell conditioned medium are shown in fig. 3A and 3B.
In fig. 3A and 3B, the positive control group (PC) was IMDM as described above. The PC and S1 groups shown in fig. 3A and 3B were grown by: on day 0, CD34+ hematopoietic stem cells were thawed and cultured with the UC-MSC feeder cells at a cell density of 2.5X 104Cells/ml were co-cultured for 12 days, and 5% umbilical cord serum/IMDM medium containing 6 cytokines and hydrocortisone was used as a positive control group (PC), or SCGM containing 6 cytokines, hydrocortisone and 4 auxiliary components was used as a group S1.
Group S3-2 was grown by: on day 0, CD34+ hematopoietic stem cells were thawed and mixed with a mixed medium (culture medium mix) having 50% (v/v) SF-UCM and 50% (v/v) fresh SCGM and containing 6 cytokines, hydrocortisone, and 4 auxiliary components at a cell density of 2.5X 104Cells/ml were co-cultured for 12 days. The 6 cytokines, hydrocortisone and 4 auxiliary components used in this example were the same as those used in example 2.
During the 12 day culture period, 50% of the medium and freshly prepared UC-MSC feeder cells were supplemented every 4 days, and the cells (including CD34+ cells) were maintained at 2.5 to 5X 104Cell density of cells/ml. On day 12, cumulative total cell expansion fold and CD34+ (ishag) expansion fold were calculated.
As shown in the experimental results of FIGS. 3A and 3B, SF-UCM was able to effectively replace the feeder layer without significantly affecting the total cell expansion. Furthermore, SF-UCM may also replace the feeder layer used in the expansion of CD34+ cells, although this is somewhat less effective. Based on the above experimental results, SF-UCM is a good substitute for feeder layers.
experimental example 4: comparison of SF-UCM and concentrated SF-UCM as alternatives to feeder layers
The following experimental examples will evaluate the effect of replacing the feeder layer with SF-UCM or concentrated SF-UCM. The SF-UCM obtained in example 3 was concentrated using a 5 kilodalton to 10 kilodalton blocking membrane (preferably a 5 kilodalton blocking membrane) to obtain a 10-fold concentrated (by volume) conditioned medium. The concentrated conditioned medium was filtered using a 0.22 μm filter, and the filtrate was collected to obtain the desired concentrated umbilical cord mesenchymal stem cell conditioned medium (abbreviated as "c-SF-UCM"). The results of experiments using SF-UCM or c-SF-UCM instead of the feeder layer are shown in FIGS. 4A and 4B.
In FIGS. 4A and 4B, the groups PC, S1 and S3-2 were the same as those described in Experimental example 3. Group S3-3 was grown by: on day 0, CD34+ hematopoietic stem cells were thawed and mixed with a mixture of 5% (v/v) concentrated SF-UCM and 95% (v/v) SCGM and containing 6 cytokines, 4 accessory ingredients, and hydrocortisoneThe cell density of the culture medium is 2.5X 104Cells/ml were co-cultured for 12 days. The 6 cytokines, hydrocortisone and 4 auxiliary components used in this example were the same as those used in example 2. During the culture period, 50% of each of the above mixed media was supplemented every 4 days, and cells (including CD34+ cells) were maintained at 2.5 to 10X 104Cell density of cells/ml. On day 12, cumulative total cell expansion fold and CD34+ (ishag) expansion fold were calculated.
As shown in the experimental results of fig. 4A and 4B, the concentrated SF-UCM was more effective than SF-UCM in supporting total cell expansion and expansion of CD34+ cells. In fact, concentrated SF-UCM is also more effective than using a feeder layer. That is, concentrated SF-UCM showed optimal stem cell expansion compared to either SF-UCM or feeder layer. The fact that concentrated SF-UCM is superior to the feeder layer is surprising. The results of these experiments indicate that specific factors have better activity in conditioned media at higher concentrations (compared to those produced by the feeder layer).
Experimental example 5: the combination of 4 auxiliary components and concentrated SF-UCM has great improvement effect on HSC expansion
As a result of the above experiment, when the feeder layer was used together with 4 kinds of auxiliary ingredients (vitamin C, vitamin E, estradiol and transferrin), the expansion effect of the stem cells was not significantly enhanced. As shown in Experimental example 4, since the concentrated SF-UCM has a better activity, the effect of using the concentrated SF-UCM (abbreviated as "c-SF-UCM") in combination with 4 kinds of auxiliary components was further tested. The experimental results of these tests are shown in fig. 5A and 5B.
On day 0, CD34+ hematopoietic stem cells were thawed and cultured in media from the following groups: (1) SCGM + SP + UCM group: a mixed culture medium having 5% (v/v) c-SF-UCM and 95% (v/v) SCGM and comprising 6 cytokines, 4 auxiliary components, and hydrocortisone; (2) SCGM + SP group: SCGM containing 6 cytokines, 4 auxiliary components and hydrocortisone; (3) SCGM + UCM group: a mixed medium having 5% (v/v) c-SF-UCM and 95% (v/v) SCGM and comprising 6 cytokines and hydrocortisone; and (4) SCGM group: SCGM with 6 cytokines and hydrocortisone. The 6 cytokines, hydrocortisone and 4 auxiliary components used in this example were the same as those used in example 2.
The cells were at 2.5X 104The cells were cultured at a cell density of cells/ml. During the culture period, 50-80% of each of the above mixed media was supplemented every 4 days, and cells (including CD34+ cells) were maintained at 2.5 to 10X 104Cell density of cells/ml. On day 12, cumulative total cell expansion fold and CD34+ (ishag) expansion fold were calculated.
Fig. 5A and 5B show the cumulative fold expansion of total cells and the fold expansion of CD34+ (ishag) compared to the SCGM group. As shown in FIGS. 5A and 5B, 4 accessory components and c-SF-UCM enhanced the expansion of total cells and CD34+ cells, respectively, in the presence of SCGM and 6 cytokines (but in the absence of a feeder layer).
The fact that 4 accessory components can enhance cell expansion was unexpected and contrary to the results seen in the presence of a feeder layer (see fig. 2A and 2B). As mentioned above, some factors secreted by the feeder layer may have effects similar to those of the 4 accessory components. Therefore, the effect of the 4 accessory ingredients on enhancing cell expansion was not significant when the feeder layer was used, but was significant when the c-SF-UCM was used instead of the feeder layer. That is, when the feeder layer is not present, 4 accessory ingredients can substitute for the missing factor, thereby producing an enhancing effect.
When 4 accessory components and c-SF-UCM were added to the same medium, the cumulative total cell expansion fold or CD34+ cell expansion fold increased significantly. The possible reason is that the 4 accessory components and factors in c-SF-UCM may contribute to different stages of the cell expansion pathway, so that their combination brings about a great improvement effect on the expansion of hematopoietic stem cells. Based on this, 4 kinds of auxiliary components and c-SF-UCM can act together in a synergistic manner.
Experimental example 6: hematopoietic stem cell expansion does not affect Colony Forming Units (CFU)
In vitro expansion of stem cells requires symmetric division, where two daughter cells retain the properties of the stem cell. One problem encountered in expanding hematopoietic stem cells in vitro is the differentiation and maturation that may be encountered when expanding the cells. The differentiated cells may not develop into the desired type of cells after transplantation.
For the detection of committed hematopoietic progenitors (committed hematopoietic progenitors), day 0 uncultured CD34+ cells and day 12 cultured CD34+ cells were seeded at a concentration of 100 and 5000 cells/ml, respectively, in 35mm dishes and cultured in cytokine-supplemented MethoCult methylcellulose Medium (Stemcell Technology, Vancouver, Canada). In a moisture saturated environment at 37 ℃ and at 20% O2And 5% CO2After 14 days of culture, total Colony Forming Units (CFUs) including CFU-G, CFU-M, CFU-GM, CFU-E and BFU-E were counted using an inverted microscope (inverted microscope). The cumulative CFU fold expansion for each group was normalized to the total number of uncultured CFUs at day 0 and shown as relative fold expansion.
As shown in fig. 6A and 6B, the cumulative CFU fold expansion parallels the cumulative CD34+ cell fold expansion (ishag), indicating that the expanded CD34+ cells maintain their stem cell characteristics.
Experimental example 7: testing the Effect of each of the 4 auxiliary ingredients
As shown in the experimental examples described above, 4 accessory ingredients enhanced cell expansion in the media of the present invention in the absence of a feeder layer. To further understand the role of the accessory ingredients, we investigated the individual roles of each accessory ingredient.
In this experimental example, the PC group and the S3-3 group were the same as those in the previous experimental example. Every 4 days, 50% of the medium was supplemented, and the cells were maintained at 2.5 to 5X 104Cell density of cells/ml.
Other groups were prepared as follows: on day 0, CD34+ hematopoietic stem cells were thawed and cultured in media from the following groups: (1) SP3 in UCM group: has 5% (v/v) C-SF-UCM and 95% (v/v) SCGM, and comprises 6 cytokines, 3 auxiliary components (estradiol E2+ vitamin C + vitamin E)) And a mixed medium of hydrogenated pinus; (2) SP3 in SCGM group: SCGM containing 6 cytokines, 3 auxiliary components (estradiol E2+ vitamin C + vitamin E) and hydrocortisone; (3) UCM group: a mixed medium having 5% (v/v) c-SF-UCM and 95% (v/v) SCGM and comprising 6 cytokines and hydrocortisone; and (4) SCGM group: SCGM with 6 cytokines and hydrocortisone. The 6 cytokines, hydrocortisone and adjunct ingredients used in this example were the same as those used in example 2. The cells were at 2.5X 104The cells were cultured at a cell density of cells/ml. During the culture period, 80% of each of the above mixed media was supplemented every 4 days, and the cells (including CD34+ cells) were maintained at 2.5 to 10X 104Cell density of cells/ml. On day 12, cumulative total cell expansion fold and CD34+ (ishag) expansion fold were calculated.
FIGS. 7A and 7B show the results of evaluation of the effect of each accessory component on the expansion of hematopoietic stem cells. The experiment detects each auxiliary component, and the experimental result shows that the use of Transferrin (TF) can be excluded. As shown in fig. 7A and 7B, the remaining 3 auxiliary components, vitamin C, vitamin E and estradiol (E2), when added, enhanced the fold expansion of cells. In contrast, further addition of Transferrin (TF) did not significantly enhance cell expansion.
fig. 8A and 8B show the results of evaluating the effect of vitamin C on hematopoietic stem cell expansion. The experimental results show that vitamin C can independently support the expansion of total cells and the expansion of CD34+ cells. Fig. 9A and 9B show the results of evaluating the effect of vitamin E on hematopoietic stem cell expansion. The experimental results show that vitamin E can also support the expansion of total cells and CD34+ cells alone. Fig. 10A and 10B show the results of evaluating the effect of vitamin C and vitamin E in combination on hematopoietic stem cell expansion. The experimental results show that the combination of vitamin C and vitamin E also gives good results in supporting cell expansion.
Experimental example 8: different amounts of media supplements (media replenishments)
The results of the above experimental examples show that in the expansion of isolated hematopoietic stem cells, serum and feeder layers in the medium can be replaced with specific factors and auxiliary components. To further investigate the mode of culture, the culture procedure was varied. The conditions of this example were the same as those of the above example, except that the amount of the supplemented (replenished) medium was changed every 4 days. One group was supplemented with 50% (1: 1) every 4 days, and the other group was supplemented with 80% (1: 4) every 4 days. The results of the experiment are shown in FIGS. 11A and 11B.
From the experimental results of fig. 11A and 11B, when different amounts of the medium were supplemented, supplementation with an amount of 80% every 4 days gave better results than supplementation with an amount of 50% every 4 days. Obviously, cells would benefit from a refreshed medium.
Experimental example 9: cytokines required for hematopoietic stem cell expansion
In the above experimental examples, 6 cytokines were included in the culture medium: rh SCF, rh TPO, rh Flt3L, rh IL-3, rh IL-6, and rh G-CSF. To test the necessity of all cytokines, the following experiments were performed to exclude some cytokines.
In this experimental example, umbilical cord mesenchymal stem cells (UC-MSC) were seeded into a T-12.5 vessel on day-1 and cultured as feeder cells in complete culture medium (containing 10% human umbilical cord serum and DMEM). On day 0, CD34+ hematopoietic stem cells were thawed and cultured with the UC-MSC feeder cells at a cell density of 2.5X 104Cells/ml were co-cultured for 6 days, and 2ml of 5% CS/IMDM containing 3, 5 or 6 cytokines and hydrocortisone were used in combination. The 3 cytokine group (cytokines x 3) included rh SCF, rh TPO, and rh Flt 3L. The 5 cytokine group (cytokines x 5) included rh SCF, rh TPO, rh Flt3L, IL-3 and IL-6. The 6 cytokine groups (cytokines x 6) included rh SCF, rh TPO, rh Flt3L, rh IL-3, rh IL-6 and rh G-CSF. During cell culture, an additional 3ml of mixed medium was added. On day 6, the cumulative total cell expansion fold and CD34+ (ishag) expansion fold were calculated.
QC group: COH Medium (15% FBS/Myelocu) was usedlt H5100+ IMDM) hematopoietic stem cells (including CD34+ cells) were co-cultured with COH275 feeder cells and 3 cytokines including rh SCF, rh TPO and rhFlt3L were used in combination. The cell density was 2.5X 104Cells/ml. 3ml of mixed medium was added on day 3 and day 5. The experimental results are shown in fig. 12A and 12B.
From the experimental results, both the expansion of total cells and the expansion of CD34+ cells benefit from more cytokines: 6 cytokines >5 cytokines >3 cytokines. However, in the expansion of total cells, the tendency to select for more cytokines is evident, whereas in CD34+ cells, this tendency is less evident. This observation indicates that non-CD 34+ cells in the total cell population would benefit from more cytokines. This phenomenon can be seen from the results of fig. 13 showing the relative expansion of CD34+ cells relative to total cell expansion. Interestingly, in this experimental example, 3 cytokines appeared to be sufficient to support the expansion of CD34+ cells, while the other extra cytokines (in the group of 5 cytokines versus 6 cytokines) appeared to be more beneficial for non-CD 34+ cells. While the use of other redundant cytokines slightly enhanced the expansion of CD34+ cells compared to the 3 cytokines.
Experimental example 10: erythroid lineage cells in expanded cells
It is known that expansion of hematopoietic stem cells ex vivo will result in some differentiated cells and some committed cells for a particular lineage. For the detection of committed hematopoietic progenitors (hematopoietic progenitors), day 0 uncultured CD34+ cells and day 12 cultured CD34+ cells were seeded at a concentration of 100 and 5000 cells/ml, respectively, in 35mm dishes and cultured in cytokine-supplemented MethoCult methylcellulose Medium (Stemcell Technology, Vancouver, Canada).
In a moisture saturated environment at 37 ℃ and at 20% O2And 5% CO2After 14 days of culture, total Colony Forming Units (CFUs) including CFU-G, CFU-M, CFU-GM, CFU-E and BFU-E were counted using an inverted microscope (inverted microscope). Percentage of erythrocyte lineage CFUIs calculated using the following formula: (BFU-E + CFU-E)/(BFU-E + CFU-E + CFU-GM + CFU-G + CFU-M) × 100%. The above abbreviations are as follows: BFU-E is burst-forming unit-red blood cell (burst-erythroid); CFU-E is a colony-forming unit-red blood cell (colony-forming unit-erythroid); CFU-GM is colony-forming unit-granulocyte, macrophage (colony-forming-macrophage); CFU-G is a colony-forming unit-granulocyte, and CFU-M is a colony-forming unit-macrophage.
As shown in fig. 14, cells expanded in the media of the invention will produce a higher percentage of erythroid lineage CFU when compared to the Positive Control (PC). These experimental results indicate that the culture medium of the present invention can support expansion of hematopoietic stem cells to produce a sufficient proportion of cells that retain the characteristics of erythroid lineage progenitor cells. However, cells expanded in the media of the invention did not produce a higher proportion of erythroid lineage CFU when compared to uncultured cells. These results suggest that during cell expansion, certain hematopoietic stem cells may differentiate into other cell lineages.
From the above experimental examples, it was found that the serum-free medium of the present invention can support ex vivo expansion of hematopoietic stem cells. The methods of the invention may comprise growing hematopoietic stem cells in any of the media of the invention comprising a basal medium, a cytokine, specific accessory ingredients, and a conditioned medium obtained from a serum-free medium for culturing umbilical cord mesenchymal stem cells.
Advantages of embodiments of the invention may include one or more of the following. Due to the lack of serum and other tissues in the medium (feeder layer), quality control of active and key components is easier to perform. The invention can avoid potential pollution or adverse immunoreaction, improve the safety of cell therapy products and easily conform to the amplification process of GMP cell production.
Although the present invention has been described with reference to the above embodiments, it should be understood that the invention is not limited thereto, and various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention.