EP4684003A2

EP4684003A2 - Method for conducting static cell culture and culture media for the differentiation of human adipose tissue stem cells into bone cells

Info

Publication number: EP4684003A2
Application number: EP24731680.5A
Authority: EP
Inventors: Anna OSYCZKA; Karolina TRUCHAN; Katarzyna CHOLEWA-KOWALSKA; Barbara ZAGRAJCZUK
Original assignee: Uniwersytet Jagiellonski; Akademia Gomiczo Hutnicza
Current assignee: Uniwersytet Jagiellonski; Akademia Gomiczo Hutnicza
Priority date: 2023-03-20
Filing date: 2024-03-20
Publication date: 2026-01-28
Also published as: WO2024196270A2; WO2024196270A3; PL444132A1; PL249233B1

Abstract

The subjects of the invention are methods for stimulating the differentiation of stem cells into bone cells in a static or dynamic cell culture. The invention also includes culture media for the stimulation and maintenance of cellular osteogenesis.

Description

Method for conducting static cell culture and culture media for the differentiation of human adipose tissue stem cells into bone cells

The subject of the invention is a method for conducting cell culture and culture media for the differentiation of human adipose tissue stem cells into bone cells.

The invention is applicable for the efficient in vitro stimulation of the differentiation of human mesenchymal stem cells (MSCs) of adipose tissue into bone cells by using a composition of chemical agents in static or dynamic culture on defined composite surfaces.

Mesenchymal stem cells (MSCs) are also referred to by the term medicinal signalling cells (MSCs; a name adopted in 2017 by Arnold Caplan [1], after modifying the previously adopted name Mesenchymal Stem Cells while retaining the same MSC abbreviation). MSCs have been used for many years to induce, among other, the bone formation processes due to the broad application potential of bone forming-initiated MSC cells in bone tissue and bone-related theapies. MSCs are found in many tissues of adult organisms, although bone marrow MSCs have been the longest studied and hence the most widely used for research and therapy. MSCs of adipose tissue also show potential for tissue regeneration and differentiation into non-fat cell types, including bone-forming potential. Since the discovery of MSCs in adipose tissue and the definition of their differentiation potential, they have immediately become a promising alternative to bone marrow MSCs in bone tissue engineering and clinical bone tissue therapies, among others. It is not clear whether adipose tissue MSCs have better, worse or the same bone-forming potential compared to bone marrow MSCs, but many literature reports suggest that adipose tissue MSCs have a weaker bone-forming potential compared to bone marrow MSCs.

To enhance the effects of differentiating adipose tissue MSCs into bone cells, various strategies of culturing and delivering them to the recipient are being developed to maximise their clinical potential.

Human recombinant bone morphogenetic protein type 2 (BMP-2) is a cell-stimulating agent known from the state of the art, approved by the U.S. Food and Drug Administration (FDA) for some clinical applications, mainly in the treatment of bone tissue. However, a number of basic studies, e.g. [2], and clinical trials suggest that BMP-2 may not induce bone formation in human MSCs as efficiently as, for example, in rodent MSC cultures. Furthermore poly(lactic-co-glycolic acid), PLGA is known clinically applicable material. It is characterised by its biocompatibility and controlled rate of biodegradation to products naturally metabolised by the body. PLGA has been approved for clinical use in biomedical products such as bioresorbable and biodegradable sutures. However, PLGA does not have bone formation-inducing (in other words osteoinductive) properties, but its modifications can increase its affinity to cells as well promote stable binding to cells and the extracellular matrix they form. We have previously demonstrated that composite materials based on PLGA modified with bioactive glasses of the group SiO₂-CaO or SiO₂-CaO-P₂O₅, obtained as growth surfaces for human bone marrow MSCs in in vitro cultures, can induce bone formation in these cells without the need to stimulate cells with additional bone formation-initiating factors [3], Nevertheless, the above composite materials are not sufficient on their own for the differentiation of adipose stem cells. Unexpectedly, the above problems have been solved in the present invention.

A first subject of the invention is a method for conducting cell culture, comprising: a) providing a multi-well vessel for cell culturing, b) placing a growth surface in at least one culture well, c) seeding human mesenchymal stem cells in the culture well with a growth surface in a first culture medium, d) conducting the cell culture, characterised by that in the step d) on the first day of culture the first culture medium is exchanged for a third culture medium, and the latter is exchanged for fresh portions of the third culture medium during the course of the culture, optionally the third culture medium is alternated with a second culture medium during the course of the culture, and the in vitro culture is carried out to achieve differentiation of mesenchymal stem cells (MSC) of adipose tissue towards bone cells, wherein the growth surface in step b) is a composite surface containing poly(lactic-co-glycolic acid), PLGA and bioactive glass particles in a weight ratio of 1:1, wherein a standard culture medium is used as the first culture medium, preferably containing 89% (v/v) MEM Alpha or DMEM/F12, 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics, while the first culture medium supplemented with 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL, beta-glycerophosphate at a concentration of 10 mM, dexamethasone at a concentration of 10^-7 M and human recombinant bone morphogenetic protein type 2 at a concentration of 100 ng/mL is used as the second culture medium, and the first culture medium supplement with 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL, beta-glycerophosphate at a concentration of 10 mM, dexamethasone at a concentration of 10^-7 M, human recombinant bone morphogenetic protein type 2 at a concentration of 2 ng/mL to 250 ng/mL, amino-3'-methoxyflavone at a concentration of 1 μM to 80 μM and the methanesulphonic salt of 3,5-diamino-6-chloro-N- [imino(phenylamino)methyl] pyrazinecarboxamide at a concentration of 0.4 μM to 50 μM is used as the third culture medium.

Preferably, in the step d) the differentiation progression of mesenchymal stem cells towards bone cells is determined by an increase in the expression level of mRNA for bone formation- related genes selected from the group comprising: the osteoprotegrin gene, the osteocalcin gene, the osteopontin gene, the type I collagen gene, the bone morphogenetic protein type 2 gene and/or the osteonectin gene, or the protein expression level of these markers or the activity of enzymes from the group of these markers, compared to a negative control.

Preferably, in the step d) the culture is carried out until mineralization of the extracellular matrix is achieved.

In a preferred embodiment of the invention, the third culture medium is replaced with a fresh portion of the third culture medium, optionally alternating with a fresh portion of the second culture medium, at a frequency of 1 to 4 days during the course of the culture.

In a further preferred embodiment of the invention, on the first day of culture the first culture medium is exchanged for the second culture medium, then on the fourth day of culture the second culture medium is exchanged for the third culture medium, and then after three days the third culture medium is exchanged for the second culture medium, and again after three days, the second culture medium is exchanged for the third culture medium, whereby the exchange of the second culture medium for the third culture medium or the third culture medium for the second culture medium is continued until the end of the culture.

In a further preferred embodiment of the invention, the moment of the third culture medium exchange for a new portion of the third culture medium, optionally the moment of alternating exchange of the third culture medium and the second culture medium, is determined either by the colour change of the pH indicator in the culture medium or by the cell growth curve/ population doubling time. The cell growth curve allows for determining the so-called population doubling time of the entire cell population. In yet another preferred embodiment of the invention, the glass particles contain silicon oxide in an amount of 40% to 80% by weight, calcium oxide in an amount of 11% to 60% by weight and/or phosphorus(V) oxide in an amount of 4% to 6% by weight.

In a further preferred embodiment of the invention, the glass particles comprise a metal oxide in an amount of 5% by weight selected from the group consisting of: strontium oxide or zinc oxide. In a still further preferred embodiment of the invention, the mineralisation of the extracellular matrix is determined by a colorimetric method. In a further preferred embodiment of the invention, the mRNA levels for bone formation-related genes, especially selected from the group comprising: the osteoprotegrin gene, the osteocalcin gene, the osteopontin gene, the type I collagen gene, the bone morphogenetic protein type 2 gene and/or the osteonectin gene, are determined by real-time PCR. Preferably, the cultures are carried out either in a static manner or in a dynamic manner with rocker mixing of the cultures. Another subject of the invention is a culture medium for the differentiation of human adipose tissue stem cells into bone cells, characterised in that it is a composition comprising:

- a standard culture medium containing 89% (v/v) MEM Alpha or DMEM/F12, 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics,

- 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL,

- beta-glycerophosphate at a concentration of 10 mM,

- dexamethasone at a concentration of 10^-7 M,

- human recombinant bone morphogenetic protein type 2 at a concentration of 100 ng/mL. Another subject of the invention is a culture medium for the differentiation of human adipose tissue stem cells into bone cells, characterised in that it is a composition comprising:

- 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL,

- beta-glycerophosphate at a concentration of 10 mM,

- dexamethasone at a concentration of 10^-7 M,

- human recombinant bone morphogenetic protein type 2 at a concentration of 100 ng/mL,

- 2'-amino-3'-methoxyflavone at a concentration of 50 μM, methanesulfonium salt of 3,5-diamino-6-chloro-N-[imino(phenylamino)methyl] pyrazinecarboxamide at a concentration of 20 μM.

The cocktail of chemical agents added to the culture medium for adipose tissue MSCs, which is one of the aspects of the invention, includes human recombinant bone morphogenetic protein type 2 (rhBMP-2) and additional factors promoting its action. It has previously been shown that the efficacy of BMP-2 in human bone marrow MSCs can be enhanced by the use of the ERK kinase inhibitor PD 98059 [4], which is a component factor in the developed chemical cocktail used to stimulate bone formation in adipose tissue MSCs. It has also been reported that the chemical compound Phenamil in adipose tissue MSC cells can induce the production of Trb3 proteins, which in turn inhibit the degradative effect of Smurfl on pSMADl/5/8 proteins - important for signal transduction by BMPs [5], Therefore, the developed innovative chemical cocktail includes the human recombinant bone morphogenetic protein type 2 - rhBMP-2, the ERK kinase inhibitor PD 98059 (2'-amino-3'- methoxyflavone, CAS: 167869-21-8) and the chemical compound Phenamil (methanesulphonic salt of 3,5-diamino-6-chloro-N-[imino(phenylamino)methyl] pyrazinecarboxamide, CAS: 1161-94-0). All the indicated agents are added to the primary osteogenic medium (i.e. MEM Alpha or DMEM/F12, bovine serum, antibiotics, 2-phospho-L- ascorbic acid, dexamethasone and beta-glycerophosphate). The component concentrations of the chemical cocktail and the culture medium are detailed in Example 1.

The invention also relates to the use of bioactive growth surfaces for cell culture obtained on the base of PLGA and bioactive glasses SiO₂-CaO or SiO₂-CaO-P₂O₅ appropriately modified with strontium oxide (SrO) or zinc oxide (ZnO). Importantly, only the use of the developed cocktail of chemical agents added to the culture medium, either in static or dynamic culture on the aforementioned bioactive surfaces, yields the expected results in the MSC cultures of adipose tissue. Furthermore, the SiO₂-CaO or SiO₂-CaO-P₂O₅ surfaces modified with SrO or ZnO further increase the expression of some mRNAs and proteins closely related to bone formation processes in adipose tissue MSCs, compared to basic surfaces.

The invention further comprises a dynamic culture - a method for mechanically enhancing the osteogenic differentiation of adipose tissue MSCs cultured on the indicated bioactive surfaces, comprising the continuous and stable mixing of the culture medium in culture plates, at well- defined rates and culture time, using a typical laboratory rocker shaker.

Using the cocktail of chemical agents allows for inducing in a short time the differentiation of human adipose MSCs into bone cells on composite surfaces. This results in increased expression of genes related to bone formation and production of bone extracellular matrix, leading to the deposition of the mineral necessary for bone formation. The composite surfaces, together with the chemical cocktail, promote the differentiation of human adipose MSCs into bone cells in culture in vitro and, at the same time, can serve as a scaffold or carrier for introducing cells to the host in vivo.

The invention, which relates to the stimulation of bone formation processes (i.e. osteogenesis) in human adipose tissue MSCs, thus comprises the cocktail of chemical agents added to the primary osteogenic medium for cells cultured in vitro on specific composite surfaces under static or dynamic (i.e. with rocker-based mixing) culture conditions. The invention enables efficient induction of adipose tissue MSCs differentiation into bone cells in vitro and delivering them in vivo to a potential recipient. It is possible to provide a patient with the chemical cocktail described in the invention and/or MSC-populated surfaces or scaffolds of the indicated composition. Thus, both the cocktail of chemical agents as well the composite surfaces and/or MSC-populated composite surfaces are possible for in vivo application (together or separately).

An unexpected result obtained according to the invention is the possibility to stimulate differentiation of the human adipose tissue mesenchymal stem cells into bone cells rapidly and completely.

In the course of the work leading to the invention, it was unexpectedly found that conducting cultures with the use of the composite surfaces modified with SrO or ZnO in the medium of the composition defined according to the invention as the third medium leads to a significant increase in the expression of bone cell-specific genes (see Example 3). Unexpectedly, the modified composite surfaces were found to promote the bone-forming processes in these cultures synergistically with the chemical cocktail.

Embodiments of the invention are illustrated in Figures 1-11.

Fig. 1. The mRNA levels of selected bone formation-related markers after (A) 7-day and (B) 21- day culture of human adipose tissue MSCs on basic (i.e. unmodified with ZnO nor SrO) composite surfaces (Al, A2, SI, S2 and PLGA basal material). Variables are cells cultured in the basic osteogenic medium - the second culture medium (grey bars) or with the chemical cocktail - the third culture medium (black bars), compositions given in Table 2. ANOVA tests, *p<0.05 compared to the adipose tissue MSCs cultured on poly(lactic-co-glycolic acid), PLGA control surface and treated only with the basic osteogenic medium (line) and between trials on each surface treated with the basic osteogenic medium or the chemical cocktail. BMP-2 - bone morphogenetic protein type 2, ON - osteonectin, OC - osteocalcin, OPG - osteoprotegrin; Fig. 2. The mRNA levels of selected bone formation-related markers in human adipose tissue MSCs after 7-day culture with the formulated chemical cocktail (the third culture medium). Variables are basal unmodified composite surfaces (white bars) and analogous composite surfaces modified with SrO (grey bars) or ZnO (black bars). ANOVA tests, *p<0.05 compared to the results on basic, unmodified composite surfaces. Line indicates mRNA expression levels for cells cultured on PLGA control surfaces. BMP-2 - bone morphogenetic protein type 2, OPG - osteoprotegerin;

Fig. 3. The mRNA levels of selected bone formation-related markers in human adipose tissue MSCs after 3-day culture with the formulated chemical cocktail on basal and SrO- or ZnO- modified composite surfaces. Static culture without rocker mixing stimulation (grey bars) and dynamic culture with rocker mixing applied throughout the culture (black bars). Culture on basal composite surfaces in culture medium containing ascorbic acid, dexamethasone and beta-glycerophosphate (white bars) was used as a control. ANOVA tests, *p<0.05 compared to the basal composites of a given type in a control sample under static conditions or between static and dynamic culture on particular surfaces. OPG - osteoprotegrin, OC - osteocalcin;

Fig. 4. The mRNA levels of selected bone formation-related markers in human adipose tissue MSCs after 7-day culture with the formulated chemical cocktail on basal, unmodified composite surfaces. The variables are static culture without rocker mixing stimulation (grey bars) and dynamic culture with rocker mixing of the cultures (black bars). The method of dynamic stimulation using rocker mixing in the cell culture is further illustrated in Fig. 10. ANOVA tests, *p<0.05 compared to the PLGA control trial under static conditions (line) or between static and dynamic culture on particular surfaces. OC - osteocalcin, OPN - osteopontin;

Fig. 5. The mRNA levels of selected bone formation-related markers in human adipose tissue MSCs after 7-day culture with the formulated chemical cocktail on SrO- or ZnO-modified composite surfaces. The variables are static culture without rocker mixing stimulation (light bars) and dynamic culture with rocker mixing of the culture (dark bars). The method of dynamic stimulation using rocker mixing in the cell culture is further illustrated in Fig. 10. ANOVA tests, *p<0.05 compared to the PLGA control trial under static conditions or between static and dynamic culture on paticular surfaces. OPG - osteoprotegrin, OC - osteocalcin;

Fig. 6. (A) The mRNA levels of selected bone formation-related markers in human adipose tissue MSCs after 21-day culture with the formulated chemical cocktail on basal unmodified composite surfaces (Al, A2, SI, S2) and analogous composite surfaces modified with SrO or ZnO. The variables are static culture without rocker mixing stimulation (grey bars) and dynamic culture with rocker mixing of the cultures (black bars). Results are shown as the expression levels relative to the PLGA control surfaces (line). The method of dynamic stimulation using rocker mixing in the 21-day cell culture is further illustrated in Fig. 10. OPG - osteoprotegerin, COL1A - type 1 collagen. (B) The level of extracellular matrix mineralisation in human adipose tissue MSCs cultured with the formulated chemical cocktail for 7 days on composite surfaces as above and then transferred from the composite surfaces to standard culture dishes and cultured as above for a further 14 days. The variables are static culture without mechanical stimulation (grey bars) and dynamic culture using rocker mixing of the culture (black bars). The mineralisation level determined by colorimetric method (described in Example 1) and normalised to the number of viable cells (MTS units). ANOVA tests, *p<0.05 compared to the given basal composite surface in static or dynamic culture, or between selected trials;

Fig. 7. Comparison of the mRNA levels for the selected bone formation-related gene in human adipose tissue MSCs after 3-day culture with the formulated chemical cocktail added to the culture medium which is composed mainly of either MEM Alpha (white bars) or DMEM/F12 (grey bars) on basal and ZnO-modified composite surfaces with/or dynamic culture using the rocker mixing method applied throughout the culture. ANOVA tests, *p<0.05 compared to the basal composite surface in static culture or between selected trials. OPG - osteoprotegerin, OC - osteocalcin;

Fig. 8. Parameters of the culture well in a 24-well plate in the rocker mixing method described in Example 3: (diameter) d = 15.5 mm, (height) h = 17.5 mm and 1 mL of medium. The tilt angle on the rocker mixer was (α) 7° and the mixing frequency of the culture fluid was 6 RPM. The above parameters are used to represent the shear stress of the culture medium potentially affecting the cultured cells;

Fig. 9. The scheme of human adipose MSCs (described in Examples 1 and 2) culture on composite surfaces, subjected to treatment with the formulated chemical cocktail from the first day of culture (top panel) or alternatively with the chemical cocktail in longer cultures exchanged with the basic osteogenic medium (bottom panel) at specific culture time points; Fig. 10. The schedule of human adipose tissue MSCs (described in Example 3) culture on composite surfaces, subjected to treatment with the formulated chemical cocktail and rocker mixing stimulation from the first day of culture (top panel) or with the formulated chemical cocktail and rocker mixing stimulation at specific culture time points (bottom panel).

Fig. 11. The mRNA levels of selected bone formation-related markers in human adipose tissue MSCs in 3-day cultures treated with the formulated chemical cocktail under rocker mixing stimulation. Dynamic culture using a rocker mixer, exactly as described in the original filing, is used throughout the culture. Fig. 11a) The variables are the concentrations of the individual components of the proprietary chemical cocktail, i.e. rhBMP-2 (2-250 ng/mL) or Phenamil (0.4-50 μM), or PD 98059 (1-125 μM). Fig. lib) The variables are the different concentrations of the chemical cocktail components, in the same proportions relative to each other, (a, b) Results shown as relative expression levels vs. a positive control (=1), i.e. cells treated with 100 ng/mL rhBMP-2, 50 μM PD 98059 and 20 μM Phenamil. A negative control (dashed line) - i.e. static culture of cells treated with basic osteogenic agents (ascorbic acid, dexamethasone and beta-glycerophosphate) - was used as an additional reference. OPG - osteoprotegerin, COL1A1 - type 1 collagen, OPN - osteopontin.

Example 1

Human adipose tissue MSCs were cultured on CaO-SiO₂/PLGA (Al, SI) or CaO-SiO₂-P₂O₅/PLGA (A2, S2) glass-polymer composite surfaces experimentally produced at the Department of Glass Technology and Amorphous Coatings of the AGH University of Science and Technology in Krakow, Poland [5, 6], The surfaces based on PLGA, poly(lactic-co-glycolic acid, (Mn = 220 kDa, lactide :glycolide ratio 85:15) were obtained by incorporating bioactive glasses CaO-SiO₂- P2O5 or CaO-SiO₂ into PLGA in an amount of 50% by weight. The bioactive glasses were obtained using the sol-gel method (sol-gel-derived bioactive glasses, SBG) with the compositions given in Table 1. These surfaces are known from the state of the art [5, 6], The average particle size does not exceed 45 pm (sieve analysis).

Table 1. Oxide compositions (molar %) of bioactive glasses used to obtain composite surfaces based on PLGA and 50% by weight of specific bioactive glass.

For cell culture, the aforementioned composite surfaces were sterilised by immersion in 70% ethanol, rinsing in phosphate buffered saline (PBS) and irradiation on each side with UVC light for 10 min. To hold the composite surfaces at the bottom of the culture wells, rings cut from sterile 15-mL NEST propylene tubes were used, corresponding to the diameter of the well of 24-well culture plate, each max. 1 cm in high. Adipose tissue MSCs were seeded at 10,000 cells/cm² in so-called standard culture medium [7] with a volume composition of: 89% MEM Alpha or DMEM/F12 (culture medium performance comparison Fig. 7), 10% fetal bovine serum (Biological Industries) and 1% antibiotics (ZellShield, Minerva Biolabs). Cells were stored in a humidified incubator at 37°C/5% CO₂.

On the first day of culture, the standard culture medium was replaced with the basic osteogenic medium (Table 2) containing the standard culture medium, 100 μg/mL of 2- phospho-L-ascorbic acid medium (CAS: 1713265-25-8), dexamethasone (CAS: 50-02-2) at a final concentration of 10^-7 M and beta-glycerophosphate (CAS: 154804-51-0) at a final concentration of 10 mM (all reagents from Merck) and human recombinant bone morphogenetic protein type 2 rhBMP-2 (Gibco) at 100 ng/mL.

The medium was replaced every three days during the culture, due to the cell population doubling time, which was 45 hours. Faster cell growth may serve as an indicator for earlier medium replacement. In order to directly determine the population doubling time, cells growth curve can be performed before the cultures described in the invention are started. Indirectly, during the culture course , if the basic, commercially available culture medium contains a dye sensitive to the pH of the medium, e.g. phenol red, the colour of the culture medium indicates the depletion of nutrients and the accumulation of metabolic products by changing the pH of the medium to acidic and changing the colour of the pH indicator. In this case, phenol red (a component of MEM Alpha and DMEM/F12 used in the Examples) changes colour from red-orange to yellow, indicating when the culture medium should be replaced.

From the fourth day of culture (every six days), the chemicals PD 98059 (2'-amino-3'- methoxyflavone; Merck) at a final concentration of 50 μM and Phenamil (Phenamil methanesulfonate salt, R&D Systems) at a final concentration of 20 μM were added to the basic osteogenic medium.

In the figures and further description, the chemical cocktail represents the basic osteogenic medium enriched with PD 98059 and Phenamil.

Adipose tissue MSCs were cultured for 3, 7 or 21 days on basal, unmodified composite surfaces. The MSC culture periods were selected with the aim to test the effects of applied elements of the invention - the chemical cocktail and/or dynamic culture (using the rocker mixing method), either once (in a 3- or 7-day culture) or repeatedly (in a 21-day culture). An indicator of the effect is a statistically significant increased level of mRNA expression for selected genes involved in bone formation processes. In the Examples, RT-qPCR analysis (2^_ ^AACT method) of mRNA levels for the osteoprotegrin gene, osteocalcin gene, osteopontin gene, type I collagen gene, bone morphogenetic protein type 2 gene and osteonectin gene was used. However, the indicated genes are examples and are not a limitation of the invention. A person skilled in the field could also refer to other genes that are expressed at the RNA level in bone formation-related processes, the level of selected bone formation-related proteins or the activity of enzymes from the group of bone formation-related proteins. The culture can be optionally carried out to obtain mineralisation of the extracellular matrix. In the provided Example, a 7-day culture of cells was carried out on composite surfaces, followed by cells transfer to a standard culture dish. Preferably, the mineralisation level can be determined by alizarin red staining of the extracellular matrix. In addition, the matrix-bound dye can be extracted using 5% perchloric acid and the colour intensity of the extracted dye measured by absorbance at 405 nm.

In both the 7-day and 21-day experiments, two parallel cultures were carried out, receiving either a fresh portion of the basic osteogenic medium - the second culture medium (control) - every three days, or alternating every three days between the basic osteogenic medium (the second culture medium) and the chemical cocktail (the third culture medium; Fig. 9, bottom panel).

The formulated chemical cocktail (Table 2) supports bone formation processes in human adipose tissue MSCs cultured in vitro on basal unmodified composite surfaces (as in Table 1) better than the basic osteogenic medium.

Table 2. Culture media compositions.

Example results:

In a 7-day culture of human adipose tissue MSCs (Fig. 1A) on basal composite surfaces, mRNA expression for the BMP-2 gene was significantly increased in cells treated with the chemical cocktail (black bars) on all composite surfaces tested vs. cells treated with basic osteogenic medium (grey bars). Similarly, mRNA expression for the osteonectin gene (ON) was significantly increased in cells treated with the chemical cocktail on surfaces Al and A2, and mRNA expression for the osteocalcin gene (OC) was significantly increased in cells treated with the chemical cocktail on all surfaces tested except S2 (statistically insignificant increase). On the other hand, in a 21-day culture of human adipose tissue MSCs (Fig. IB) on basal composite surfaces, mRNA levels for the BMP-2 gene were higher in cells treated with the chemical cocktail on all surfaces tested except SI (statistically insignificant increase), and mRNA expression level for the osteoprotegerin (OPG) gene, produced by bone-forming osteoblasts, were increased in cells treated with the chemical cocktail on all surfaces tested. Besides the fact that increased OPG expression indicates the differentiation of MSC cells into bone- forming-osteoblasts, OPG is a soluble receptor that binds to the RANK ligand (RANKL). The binding of OPG to RANKL blocks its interaction with the RANK receptor present on osteoclasts, the bone-resorbing cells, and prevents the RANK-RANKL interaction responsible for the osteoclast differentiation and activation. This way OPG induces osteoclast apoptosis and it is therefore a potent inhibitor of bone resorption by osteoclasts.

Furthermore, the mineralisation of the extracellular matrix after stimulation with the chemical cocktail was tested in cultures of adipose tissue MSC cells on basal composite surfaces. After 7 days of culture on composite surfaces, cells were detached from the surfaces with 0.25% trypsin/EDTA, transferred to standard culture dishes and continuously stimulated with the chemical cocktail. Mineralisation of the extracellular matrix of the cultured adipose tissue MSCs was achieved on each composite surface (Fig. 6), yet cells cultured on the S2 surface showed the highest mineralisation levels. This indicates an efficiently completed osteogenesis process.

Example 2

Human adipose tissue MSCs were cultured on composite surfaces further enriched with SrO or ZnO modification.

Bioactive glasses from CaO-SiO₂-P₂O₅-SrO/ZnO or CaO-SiO₂-SrO/ZnO systems were prepared using the sol-gel method (sol-gel-derived bioactive glasses, SBG), with a 5%mol content of SrO/ZnO oxides (glass compositions are given in Table 2). The starting materials used to obtain the glasses were: tetraethylorthosilicate Si(OC₂H₅)4 (TEOS), triethylphosphate OP(OC₂H₅)3, calcium nitrate Ca(NO₃)₂*4H₂O, zinc nitrate Zn(NO₃)₂*6H₂O, strontium nitrate Sr(NO₃)₂. The syntheses were carried out in the environment of ethanol (C₂H₅OH, 96%), water and hydrochloric acid (HCI) as a catalyst for hydrolysis and polycondensation processes. The starting solutions were left to gellify under ambient conditions and then the resulting gels were dried and heat-treated to 700°C (electric oven, heating time 20 hours). Analogously to the basal composites, the obtained glass powders with a grain size <45 pm were soaked in dichloromethane (CH2CI), which is the solvent poly(lactic-co-glycolic acid), PLGA. Solutions were prepared at the bioactive glass: polymer: solvent weight ratio of 1: 1: 53. Growth surfaces were prepared at a weight ratio of 50% bioactive glass per dry weight of PLGA copolymer using a solution casting technique. The composite surfaces were dried under cover, at ambient conditions (20°C) and then in a vacuum dryer at 800 mbar.

Table 3. Oxide compositions (%mol) of bioactive glasses used to obtain basal and SrO- or ZnO- modified composite surfaces based on PLGA and 50% by weight of specific bioactive glass.

Bioactive Content of specific oxides [%mol] in the bioactive glass glass SiO₂ P2O5 CaO SrO ZnO

Al 40 — 60

AlSr5 40 — 55 5

AlZn5 40 — 55 — 5

A2 40 6 54

A2Sr5 40 6 49 5

A2Zn5 40 6 49 — 5

SI 80 — 20 S1Sr5 80 — 15 5 S1Zn5 80 — 15 — 5

S2 80 4 16

S2Sr5 80 4 11 5

S2Zn5 80 4 11 — 5

Cells were seeded as described in Example 1 onto basal composite surfaces and analogously onto the ones modified with SrO or ZnO. Cells were treated with the chemical cocktail from the first day of culture until a significant increase in mRNA expression for bone formation- related genes, as shown in the top panel of Figure 9 (Fig. 3), or cells were treated with the basic osteogenic medium alternated with the chemical cocktail as described in the bottom panel of Figure 9 (Fig. 2). The time of the apparent increase in mRNA expression for osteogenesis-related genes can vary depending on individual characteristics or the age of the cell donor, thus the culture with the chemical cocktail administered from the first day of culture can be extended using the schedule of chemical cocktail exchange every 1-4 days.

Example results:

After 7-day culture of adipose tissue MSCs treated with the chemical cocktail, mRNA expression for the BMP-2 gene and the osteoprotegerin (OPG) gene was significantly higher on SrO or ZnO-modified composite surfaces compared to the corresponding basal composite surfaces (Fig. 2).

Furthermore, application of the chemical cocktail from day one throughout the 3-day culture of adipose tissue MSCs on SrO or ZnO oxide-modified composite surfaces (static culture) significantly increased mRNA expression for the OPG gene and the osteocalcin (OC) gene (Fig. 3).

Thus, the modified composite surfaces (as in Table 3) promote the bone-forming processes in these cultures synergistically with the chemical cocktail.

Example 3

Dynamic culture of human adipose tissue MSCs was achieved by strictly defined mixing of the culture medium at specific speeds and for specific culture times. A Biosan MR-1 rocker shaker with a tilt angle of 7° was used for this (Fig. 8). The oscillation frequency was 6 rpm. Cells were cultured on basal and ZnO- or SrO-modified composite surfaces placed at the bottom of 15.5 mm diameter wells of a 24-well culture plate, held at the bottom of the wells with rings as described in Example 1. Cells were seeded onto all tested composite surfaces and stimulated with the chemical cocktail as described in Examples 1 and 2. The total volume of culture medium was 1 mL/well of a 24-well culture plate. Dynamic cell culture was used from the first culture day throughout the culture (Fig. 3), on days 4-7 of culture (Fig. 4) or in longer cultures on the days listed in Fig. 10 in the bottom panel (Figs. 5 and 6). The rocker shaker was placed in a typical Sanyo humidified CO₂ cell culture incubator.

Example results:

Treatment of human adipose MSCs with the chemical cocktail in dynamic culture (described above) from day one, throughout the 3-day culture period, increased the mRNA expression of anayzed bone formation-related genes (Fig. 3). Dynamic culture on A2 and S2 materials modified with SrO or ZnO further increased significantly the mRNA expression level for the osteoprotegerin gene (OPG). Use of dynamic culture on days 4-7 of cell culture treated with chemical cocktail (Fig. 4), increased the expression of mRNA for the osteocalcin (OC) gene on day seven culture on all tested basal composite surfaces and the expression of mRNA for the osteopontin (OPN) gene on the Al and A2 composite surfaces. Thus, dynamic cell culture can promote bone formation processes synergistically with the chemical cocktail in short-term cell cultures on basal composite surfaces.

Dynamic culture of cells on ZnO- or SrO-modified composite surfaces treated with the chemical cocktail (Fig. 5) increased the mRNA expression level for the osteoprotegerin (OPG) gene on the A2-SrO, S1-SrO, A2-ZnO, S1-ZnO and S2-ZnO surfaces and the mRNA expression level for the osteoclactin (OC) gene on the A2-SrO, S2-SrO and S2-ZnO surfaces on day seven culture. Thus, dynamic cell culture can promote bone formation processes synergistically with the chemical cocktail in short-term cell cultures on ZnO- or SrO-modified composite surfaces. In a 21-day culture of human adipose tissue MSCs treated with the chemical cocktail on basal or SrO- or ZnO-modified composite surfaces, the introduction of dynamic culture (i.e. three times throughout the culture, as described in Fig. 10) resulted in increased mRNA for the OPG gene on the ZnO-modified surfaces and mRNA for the COL1A1 gene both on the ZnO-modified surfaces, as well as the A2-SrO and S2-SrO surfaces (Fig. 6A).

Furthermore, the effect of 7-day culture of human adipose MSCs on basal and modified composite surfaces on extracellular matrix mineralisation after a further 14 days of culture of these cells in standard culture dishes was studied. The cells were treated with the chemical cocktail as described in Example 1 and subjected to dynamic culture as shown in Fig. 10. After 7-day culture on the composite surfaces, cells were detached from the surfaces with 0.25% trypsin/EDTA, transferred to standard culture dishes and continuously stimulated with the chemical cocktail and dynamic culture as scheduled previously (Fig. 10). After a total of 21 days of cell culture (7 days of culture on composite surfaces, 14 days in standard culture dishes), the level of extracellular matrix mineralisation, normalised to the number of viable cells (Fig. 6B), was increased for: 1) static cultures on SrO-modified composite surfaces; 2) dynamic cultures on the SrO-modified Al and A2 surfaces and the ZnO-A2 and ZnO-S2 surfaces. Thus, the obtained results indicate a long-term beneficial effect of stimulating human adipose tissue MSCs with the chemical cocktail, in combination with dynamic culture. Importantly, a short-term 7-day cells culture on composite surfaces already directs the cells towards osteogenesis, which is enhanced in further culture steps by the chemical cocktail and dynamic culture. The number of viable cells (for normalisation of mineralisation levels) was determined using Promega's commercially available CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) Cat. no. G3582.

Example 4

Concentration ranges of the formulated chemical cocktail (i.e. the third medium, Table 2) positively affecting the osteogenic differentiation of adipose tissue MSC cells were determined. Human adipose tissue MSCs were seeded at 10,000/cm² in standard culture medium (the first medium, Table 2) in a 24-well culture plate. After 24 h, the above medium was exchanged for the chemical cocktail (the third medium) with different concentrations of each chemical cocktail component relevant to the invention, i.e. rhBMP-2 (2-250 ng/mL), Phenamil (0.4-50 μM), PD 98059 (1-125 μM). Both the effect of changing the concentrations of each factor in combination with the other factors at unchanged concentrations (Fig.11a) and the effect of all factors together with proportionally decreased or increased concentrations (Fig. lib) were studied. The third medium of concentrations given in Table 2 was used as a positive control (results are presented as relative expression levels of selected genes compared to positive control=l). A static culture treated with basic osteogenic agents (ascorbic acid, dexamethasone and beta-glycerophosphate) was used as a negative control. The culture was carried out for 3 days in the third medium under dynamic conditions (see Example 3 of the invention filing for a description of the dynamic culture). Indicators of the effects were statistically significant increases of mRNA expression levels for genes involved in bone formation processes. In the example, RT-qPCR analysis (2^-ΔΔCT method) of mRNA levels for osteoprotegerin (OPG), type I collagen (COL1A1) and osteopontin (OPN) was used. The genes analysed are exemplary and are not a limitation of the invention used herein.

In 3-day dynamic cultures of human adipose tissue MSCs (Fig. 11) treated with different concentrations of the chemical cocktail components (the third medium), high mRNA expression for osteoprotegerin (OPG) persists for concentrations of 2-250 ng/mL of rhBMP-2 and 0.4 μM Phenamil (Fig. 11a), as compared to cells treated with the basic osteogenic agents (dashed line). On the other hand, expression of mRNA for type 1 collagen (COL1A1) remained at increased levels for PD 98059 concentrations of 1-80 μM (Fig. 11a), as compared to cells treated with the basic osteogenic agents (dashed line). When proportionally lower (i.e. 2 ng/mL of BMP-2, 0.4 μM Phenamil, 1 μM PD 98059) or higher (i.e. 150 ng/mL of BMP-2, 30 μM Phenamil, 75 μM PD 98059) concentrations of all relevant components of the chemical cocktail were used together as compared to those described in the invention (Fig. lib), increased levels of mRNA expression for osteoprotegerin (OPG), type 1 collagen (COL1A1) and osteopontin (OPN) were observed for lower concentrations and increased levels of mRNA expression for OPG and OPN genes were observed for higher concentrations, as compared to cells treated with the basic osteogenic agents (dashed line).

Literature:

1. Caplan Al. Mesenchymal Stem Cells: Time to Change the Name! Stem Cells Transl Med. 2017 Jun;6(6):1445-1451. doi: 10.1002/sctm.17-0051. Epub 2017 Apr 28.

2. Osyczka AM, Diefenderfer DL, Bhargave G, Leboy PS. Different effects of BMP-2 on marrow stromal cells from human and rat bone. Cells Tissues Organs. 2004;176(l- 3):109-19. doi: 10.1159/000075032.

3. Lukowicz K, Zagrajczuk B, Nowak A, Niedzwiedzki L, Laczka M, Cholewa-Kowalska K, Osyczka AM. The role of CaO/SiO₂ ratio and P2O5 content in gel-derived bioactive glass-polymer composites in the modulation of their bioactivity and osteoinductivity in human BMSCs. Mater Sci Eng C Mater Biol Appl. 2020 Apr;109:110535. doi: 10.1016/j.msec.2019.110535. Epub 2019 Dec 9.

4. Osyczka AM, Leboy PS. Bone morphogenetic protein regulation of early osteoblast genes in human marrow stromal cells is mediated by extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling. Endocrinology. 2005 Aug;146(8):3428-37. doi: 10.1210/en.2005-0303. Epub 2005 May 19.

5. Fan J, Im CS, Guo M, Cui ZK, Fartash A, Kim S, Patel N, Bezouglaia O, Wu BM, Wang CY, Aghaloo TL, Lee M. Enhanced Osteogenesis of Adipose-Derived Stem Cells by Regulating Bone Morphogenetic Protein Signaling Antagonists and Agonists. Stem Cells Transl Med. 2016 Apr;5(4):539-51. doi: 10.5966/sctm.2015-0249. Epub 2016 Mar 8.

6. Pamula E, Kokoszka J, Cholewa-Kowalska K, Laczka M, Kantor L, Niedzwiedzki L, Reilly GC, Filipowska J, Made] W, Kolodziejczyk M, Tylko G, Osyczka AM. Degradation, bioactivity, and osteogenic potential of composites made of PLGA and two different sol-gel bioactive glasses. Ann Biomed Eng. 2011 Aug;39(8):2114-29. doi: 10.1007/S10439-011-0307-4. Epub 2011 Apr 13. 7. Stanners CP, Eliceiri GL, Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52-4. doi: 10.1038/newbio230052a0.

Claims

1. A method of cell culture comprising: a) providing a multi-well vessel for cell culturing, b) placing a composite surface in at least one culture well, c) seeding human adipose tissue mesenchymal stem cells onto the composite surface in a culture well in a first culture medium, d) conducting the cell culture, characterised in that in the step d) on the first day of culture the first culture medium is exchanged for a third culture medium, which is exchanged for fresh portions of the third culture medium during the course of the culture, optionally the third culture medium is alternated with a second culture medium during the course of the culture, and the in vitro culture is carried out to achieve differentiation of mesenchymal stem cells (MSC) of adipose tissue towards bone cells, wherein the culture surface in step b) is a composite surface containing lactic and glycolic acids copolymer and bioactive glass particles in a weight ratio of 1:1, wherein a standard culture medium is used as the first culture medium, preferably containing 89% (v/v) MEM Alpha or DMEM/F12, 10% (v/v) fetal bovine serum, 1% (v/v) antibiotics, whereas the first culture medium supplemented with 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL, beta-glycerophosphate at a concentration of 10 mM, dexamethasone at a concentration of 10^-7 M and human recombinant bone morphogenetic protein type 2 at a concentration of 100 ng/mL is used as the second culture medium, and the first culture medium supplement with 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL, beta-glycerophosphate at a concentration of 10 mM, dexamethasone at a concentration of 10^-7 M, human recombinant bone morphogenetic protein type 2 at a concentration of 2 ng/mL to 250 ng/mL, amino-3'-methoxyflavone at a concentration of 1 μM to 80 μM and the methanesulphonic salt of 3,5-diamino-6-chloro-N- [imino(phenylamino)methyl] pyrazinecarboxamide at a concentration of 0.4 μM to 50 μM is used as the third culture medium.

2. The method of claim 1, characterised in that in the step d) the differentiation progression of adipose tissue mesenchymal stem cells towards bone cells is determined by the increase in expression levels of mRNA for bone formation-related genes selected from the group comprising: the osteoprotegrin gene, the osteocalcin gene, the osteopontin gene, the type I collagen gene, the bone morphogenetic protein type 2 gene and/or the osteonectin gene, or the protein expression level of these markers or the activity of enzymes from the group of these markers, compared to a negative control.

3. The method of claim 1, characterised in that in the step d) the culture is carried out until mineralization of the extracellular matrix is achieved.

4. The method of claim 1, characterised in that in the step d) the third culture medium is replaced with a fresh portion of the third culture medium, optionally alternating with a fresh portion of the second culture medium, at a frequency of 1 to 4 days during the course of the culture.

5. The method of claim 1, characterised in that the first culture medium additionally contains a pH indicator, preferably phenol red.

6. The method of claim 1, characterised in that the moment of the third culture medium exchange for a new portion of the third culture medium, optionally the moment of alternating exchange of the third culture medium and the second culture medium, is determined either by the colour change of the pH indicator in the culture medium or by the cell growth curve.

7. The method of claim 1, characterized in that the glass particles contain silicon oxide in an amount of 40% to 80% by weight, calcium oxide in an amount of 11% to 60% by weight and/or phosphorus(V) oxide in an amount of 4% to 6% by weight.

8. The method of claim 1, characterized in that the glass particles comprise a metal oxide in an amount of 5% by weight selected from the group consisting of: strontium oxide or zinc oxide.

9. The method of claim 1, characterized in that the mineralisation of the extracellular matrix is determined by a colorimetric method.

10. The method of claim 1, characterized in that the mRNA level for bone formation-related genes is determined by real-time PCR.

11. The method of claim 1, characterized in that the cultures are carried out either in a static manner or in a dynamic manner with rocker mixing of the cultures.

12. A medium for the differentiation of human adipose mesenchymal stem cells into bone cells, characterized in that it is a composition comprising:

- 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL,

- beta-glycerophosphate at a concentration of 10 mM,

- dexamethasone at a concentration of 10^-7 M,

- human recombinant bone morphogenetic protein type 2 at a concentration of 100 ng/mL.

13. A medium for the differentiation of human adipose mesenchymal stem cells into bone cells, characterized in that it is a composition comprising:

- 2-phospho-L-ascorbic acid at a concentration of 100 μg/mL,

- beta-glycerophosphate at a concentration of 10 mM,

- dexamethasone at a concentration of 10^-7 M,

- human recombinant bone morphogenetic protein type 2 at a concentration of 2 ng/mL to 250 ng/mL,

- 2'-amino-3'-methoxyflavone at a concentration of 1 μM to 80 μM, methanesulfonium salt of 3,5-diamino-6-chloro-N-[imino(phenylamino)methyl] pyrazinecarboxamide at a concentration of 0.4 μM to 50 μM.

SUBSTITUTE SHEET (RULE 26)