WO2016067306A1 - Procédé de traitement de cellules nourricières adaptées à la prolifération de cellules souches adultes - Google Patents

Procédé de traitement de cellules nourricières adaptées à la prolifération de cellules souches adultes Download PDF

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WO2016067306A1
WO2016067306A1 PCT/IN2015/000404 IN2015000404W WO2016067306A1 WO 2016067306 A1 WO2016067306 A1 WO 2016067306A1 IN 2015000404 W IN2015000404 W IN 2015000404W WO 2016067306 A1 WO2016067306 A1 WO 2016067306A1
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cell
per
cells
mitomycin
feeder
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Lakshmana Kumar YERNENI
Man Rishi Chugh
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Indian Council Of Medical Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • C12N5/063—Kereatinocyte stem cells; Keratinocyte progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts

Definitions

  • This invention relates to a method for processing of feeder cells suitable for adult stem cell proliferation.
  • This invention further relates to a cost-effective yet efficient processing of feeder cells in a way that their life span and growth supporting capability are modulated through induction of controlled growth arrest in them following treatment with mitomycin C at permutations of (1) concentration per se in the treating solution and (2) logical dose amount available to the exposed cell population.
  • This invention also relates to using an optimized substratum consisting of feeder cells processed as above for culturing adult and embryonic stem cells for probable exploitation towards production of cost-effective organs for transplantation to treat certain ailments.
  • a feeder cell layer consisting usually of growth arrested fibroblast cells like the mouse embryonic skin fibroblast cells.
  • autologous keratinocytes obtained from small uninvolved skin biopsy specimen from burned patients are cultured in vitro in presence of such feeder cells that are growth arrested by exposure to gamma- irradiation and the ensuing epidermal sheets constructed in vitro are used for autologous grafting in severely burned patients (Rheinwald and Green, 1975, Atiyeh Costagliola 2007).
  • the growth arrest is reported to push the feeder cells permanently into a non-proliferative state and keep them metabolically active for a finite time period to impart growth stimulatory influence on target cells (Roy et al 2001 ; Nieto et al 2007). Alternately, exposure to other cell proliferation- blocking agents like Mitomycin C is used to growth arrest feeders as a cost effective strategy (Navasaria et al 1994).
  • a feeder cell growth arrest protocol would then consist of exposure to such derived volume-concentration permutations and generate a series of feeder cell batches from which an optimally performing batch will be identified in terms of maximal stimulation of adult stem cell proliferation.
  • This strategy is proposed to cut down the cost of culture as compared to gamma irradiation technique without compromising with culture time and target cell characteristics.
  • Another object of this invention is to propose a method for processing of feeder cells for adult stem cell proliferation, which does not employ harsh chemicals, reagents or agents.
  • Another object of this invention is to propose a method for processing of feeder cells for adult stem cell proliferation, which stimulates maximal growth of epidermal keratinocyte stem cells in a co-culture system and is comparable with gamma-irradiated (y-Irr) feeder cells.
  • Table 1 Outcome of exposure of Swiss 3T3 cells to a 2-hour pulse of various mitomycin C doses showing their relationship with the respective concentration, exposure cell density and correlation coefficient of cell number with time.
  • Exposure cell densities employed are Low (0.7276 ⁇ 0.0844 x 10 4 per cm 2 ), Medium (3.0711 ⁇ 0.3021 x 10* per cm ⁇ ) and High (6.1556 ⁇ 0.2309 x 10 4 per cm 2 ).
  • Table 3 Growth area measurement by quantitative image analysis of keratinocyte colonies grown by co-culturing 170 keratinocytes with various Swiss 3T3 feeders and colonies stained red with Rhodamine B
  • Table 4 Growth area measurement by quantitative image analysis of keratinocyte colonies grown by co-culturing 340 keratinocytes with various Swiss 3T3 feeders and colonies stained red with Rhodamine B
  • Figure 1 depicts schematic representation of experimental derivation of doses from a given concentration of Mitomycin C by cell density titrations in Swiss 3T3 cells.
  • Figure 2 shows schematic representation of producing differential growth arrest by titrations of Swiss 3T3 cells with permutations of concentrations and those doses that were experimentally derived by a process shown in Figure 1.
  • Figure 3 shows schematic representation of demonstrating the growth stimulating potential of feeder substratums prepared by a process as in Figure 2, on epidermal keratinocyte cells.
  • Figure 4 shows influence of various concentrations of mitomycin C on cell extinction/ proliferation of Swiss 3T3 fibroblast cells at various exposure cell densities.
  • the cells at Low (0.7276 ⁇ 0.0844 x 10 4 per cm 2 ), Medium (3.071 1 ⁇ 0.3021 x 10 4 per cm 2 ) and High (6.1556 ⁇ 0.2309 x 10 4 per cm 2 ) cell densities were exposed to mitomycin C at concentrations of 1 g/ml (A), 3 ⁇ g/ml (B), 4 ⁇ g/ml (C), 4 ⁇ g/ml (D) 8s 10 ⁇ g/ml (E) and periodical viable cell counts were performed on post exposure days of 3,6,9, 12 and 20.
  • Figure 5 is a scatter plot showing the four categories of trends of cell extinction /proliferation of Swiss 3T3 fibroblast cells over a 20 days period following a two-hour pulsed exposure to various mitomycin C doses/cell.
  • Figure 6 shows viable Swiss 3T3 cells recovered from T25 flasks after 2- hour pulsed exposure to mitomycin C solution having concentrations of 4Mg/ml (A) and 5 ⁇ g/ ⁇ (B), each of which was formulated to give dose permutations of 15, 75, 150 or 450 pg/cell. Each permutation was compared with concentrations of 3 and 10 g/ml, constituted at doses of 10 and 30 pg/cell, respectively, and included as the least and the most toxic permutations. Statistical comparisons performed by Student's t test were indicated as significant at p ⁇ 0.05 (*) or insignificant (NS).
  • ECN Pre-exposure cell number
  • Figure 7 shows differential periodic cell extinctions of Swiss 3T3 cells re- plated into triplicate wells of 24-well plates at a seeding density of 7,000 cells per cm 2 following a 2-hour pulsed exposure to mitomycin C in T25 flasks at concentrations of 4 ⁇ g/ml (A & B) and 5ug/ml (C 85 D), each of which was formulated to give dose permutations of 15, 75, 150 or 450 pg/cell. Viable cell counts from all the dose permutations at each time point (A 85 C) were analysed by one way ANOVA and the P value was indicated if it was less than 0.05.
  • the dose dependent variation in cell extinctions for each concentration (B 8s D) were represented by linear trend lines and R 2 values were calculated by regression.
  • Figure 8 shows comparative cellularity of Swiss 3T3 cells on days 6 (upper row) and 12 (lower row) after they were re-plated into 24-well plates following a 2-hour pulsed exposure to mitomycin C in T25 flasks at a concentration of 4 ⁇ g/ml > formulated in a way to give dose permutations of 15 (A & B), 150 (C & D) or 450 (E & F) pg/cell. Vacuolated cells (Arrows) after 6 days were rare in feeders of 4- 15 (A), few in 4-150 (C) and numerous in 4-450 (E). Overall cellularity on culture surface after 12 days post-treatment was fair in 4- 15 (B), moderate in 4- 150 (D) and poor in 4-450 (F). Marker length is 10 ⁇ .
  • Figure 9 is a clustered column diagram showing differential periodic cell extinctions of Swiss 3T3 cells re-plated at a density of 7,000 cells per cm 2 into triplicate wells of 24-well plates following a 2-hour pulsed exposure to mitomycin C in T25 flasks at concentrations, 4 ⁇ g/ml (A) and 5 ⁇ g/ml (B), each of which was formulated to give dose permutations of 15, 75, 150 or 450 pg/cell. Each permutation was compared with concentrations of 3 and ⁇ g/ml, used at doses of 10 and 30 pg/cell, respectively, and included as the least and the most toxic permutations. Each cluster represents viable cell number from a single dose after 3, 6, 9 and 12 post-treatment days. The clusters of 3- 10 and 10-30 were compared with each of the other dose permutations by paired t test and indicated as significant at p ⁇ 0.05 (*) or insignificant (NS).
  • Figure 10 shows Colony Forming Efficiency of 3 rd passage keratinocytes co-cultured with various Swiss 3T3 feeders, growth arrested by a 2-hour pulsed exposure to 4 ⁇ g (A 85 C) and 5 ⁇ g (B & D) mitomycin C per millilitre, each of which was constituted to give dose permutations of 15, 150 or 450 pg/cell.
  • Figure 11 illustrates complete color separation in a Rhodamine B stained culture plate containing keratinocyte colonies over a substratum of feeder cells, using Adobe Photoshop that facilitated quantitative image analysis.
  • the sub title of riginal' on the extreme left represents the untreated image of a Rhodamine B stained culture plate depicting keratinocyte growth area in red and feeder substratum in bluish-gray blue.
  • the sub-titles of 'Keratinocytes' and 'Feeder' represent the replicated samples of the original image from which the area in red representing the keratinocyte growth area and the remaining feeder substratum in bluish-grey, respectively, are isolated to facilitate quantification of both the areas in terms of pixels.
  • the sub-title of 'Merge' on the right shows the reconstructed combined image produced by the superimposition of 'Keratinocytes' and 'Feeder' images, to validate color isolation.
  • Figure 12 shows colony forming efficiency (A) and growth area analysis (B 85 C) of keratinocytes co-cultured with Swiss 3T3 feeders which were growth arrested by a 2-hour pulsed exposure to 4 ⁇ g mitomycin C per millilitre, constituted to give dose permutations of 15 or 150 pg/cell.
  • Feeder cells, growth arrested by gamma irradiation ( -Irr) serving as standard technique were included for comparison. Cultures were initiated in quadruplicate wells of 6-well plates and each well was seeded with 170 viable keratinocytes from frozen stock of 1 st passage cultures and 144,000 feeder cells. Colonies were counted after staining the wells with Rhodamine B (D).
  • Average colony size (C) was derived by dividing the total growth area (B) by total number of colonies (A). Significant comparison (p ⁇ 0.01) with 4- 150 feeder group is indicated by black asterisk, while comparison between feeders of 4- 15 and gamma irradiation is indicated by grey asterisk.
  • Figure 13 represents growth area analysis of keratinocytes co-cultured with Swiss 3T3 feeders which were growth arrested by a 2 -hour pulsed exposure to 4 ⁇ g mitomycin C per millilitre, constituted to give dose permutations of 15 or 150 pg/cell.
  • Feeder cells growth arrested by gamma irradiation (/-Irr) served as standard control.
  • Cultures were initiated in quadruplicate wells of 6-well plates, and each well was seeded with 340 viable 1 st passage keratinocytes and 144,000 feeder cells. Keratinocyte colonies were stained red with Rhodamine B and subjected to image analysis to calculate per cent of keratinocyte growth area from the ratio of red pixels and total number of pixels of the well.
  • the area of keratinocyte growth was calculated by considering actual total area of the well that equals to 9.6 cm 2 . 4- 15 and gamma irradiation were statistically compared with 4- 150 group by Student's t test and indicated as significant at p ⁇ 0.01.
  • Figure 14 includes Dot plots depicting distribution of cellularity (Top) BrdU positive cells (Bottom) across all keratinocyte colonies produced out of 210 plated 1 st passage keratinocytes per feeder group.
  • Feeder cells included 4- 150, 4- 15 which were growth arrested by a 2-hour pulsed exposure to 4 ⁇ g mitomycin C per millilitre, that was constituted to give dose permutations of 15 or 150 pg/cell and also the gamma irradiated feeders ⁇ -Irr). Significance (P ⁇ 0.03) is indicated by asterisk.
  • Figure 15. Pie diagram showing overall BrdU positive & negative keratinocytes with their corresponding percentages in cultures set up over feeder cells that were processed by different approaches.
  • Feeder cells included 4- 150, 4- 15 which were growth arrested by a 2-hour pulsed exposure to 4 ⁇ g mitomycin C per millilitre, that was constituted to give dose permutations of 15 or 150 pg/cell and also the gamma irradiated feeders (y -Irr). Significant (p ⁇ 0.01) comparisons with 4- 150 are indicated by white asterisk.
  • Figure 16 shows labelling of Bromodeoxy Uridine (BrdU) in human epidermal keratinocytes grown in the presence of mitomycin C feeders of 4-15 (A & B) and 4-150 groups (C & D).
  • the BrdU positive nuclei are visualized as blue-green (A & C) which are merged with corresponding phase contrast images (B & D).
  • Magnification bar 10 ⁇ .
  • Figure 17 is a clustered column diagram showing differential periodic cell extinctions of Swiss 3T3 cells re-plated at a density of 15,000 cells per cm 2 into triplicate wells of 24-well plates following a 2-hour pulsed exposure to mitomycin C in T25 flasks at concentrations, 4 ⁇ g/ml (A & C) and 5 ⁇ / ⁇ (B & D), each of which was formulated to give dose permutations of 15, 150 or 450 pg/cell.
  • the gamma irradiated feeders were represented by ⁇ -Irr.
  • Those graphs showing extinction of 7,000 plated feeders per cm 2 (B &D) are taken from Figure 9.
  • Each cluster represents viable cell number from a single dose after 3, 6, 9 and 12 post-treatment days.
  • the inter group comparisons between any two permutations were performed by paired t test and indicated as significant at p ⁇ 0.05 (*) or insignificant (NS).
  • Figure 18 is a clustered column diagram showing the periodical growth patterns of human epidermal keratinocytes grown in presence of MMC feeders of 15, 150 and 450 pg per cell under concentrations of 4 (Top) and 5 (Bottom) ⁇ g per ml and compared with those of 3- 10 and 10-30 feeders.
  • the statistical comparisons between two independent feeder cell groups were performed using cell counts from all time points by two-way ANOVA with P ⁇ 0.05 as insignificant (NS).
  • Figure 19 is a clustered column diagram showing the periodical growth patterns of human epidermal keratinocytes grown in presence of MMC feeders of 15, 150 and 450 pg per cell under concentrations of 4 (Top) and 5 (Bottom) ⁇ g per ml and compared with those of ⁇ -Irr feeders.
  • the statistical comparisons between two independent feeder cell groups were performed using cell counts from all time points by two-way ANOVA with P ⁇ 0.05 as insignificant (NS).
  • Figure 20 is a clustered column diagram showing the periodical growth output of human epidermal keratinocytes grown in presence of controls consisting of MMC feeders of 3- 10 and 10-30 and ⁇ -lrr feeders. The statistical comparisons between two independent feeder cell groups were performed using cell counts from all time points by two-way ANOVA with P ⁇ 0.05 as insignificant (NS).
  • Figure 21 shows duplicate sheets of cultured epithelia produced by confluent cultures of human keratinocytes grown over feeders of 4-15 (Top row), 4- 150 (Middle row) and ⁇ -Irr (Bottom row). The epithelia were isolated 12 days after initiating with 800 Keratinocytes/ cm 2 by incubating in dispase. Magnification: Original.
  • Figure 22 shows Hemotoxylin and Eosin stained paraffin sections of cultured epithelia produced by confluent cultures of human keratinocytes grown over feeders of 4-15 (Top row), 4- 150 (Middle row) and ⁇ -Irr (Bottom row) .
  • Figure 23 shows immuno-histochemical positive control images on localization of cytokeratin 14 (CK- 14), Involucrin, CK- 10 and Filaggrin in human epidermis.
  • the nuclei are stained blue with Dapi and the green deposits represent FITC tagged secondary antibody which was bound to the corresponding primary antibody.
  • Figure 24 shows immuno-histochemical localization of cytokeratin 14 in cultured epithelia of 4- 15 (Top), 4- 150 (middle) and ⁇ -Irr (Bottom). The nuclei are stained blue with Dapi and the green deposits represent FITC tagged secondary antibody which was bound to the an ti-cyto keratin 14 primary antibody. The left panel is the comparable Phase contrast image.
  • Figure 25 shows immuno-histochemical localization of Involucrin in cultured epithelia of 4-15 (Top), 4- 150 (middle) and ⁇ -Irr (Bottom). The nuclei are stained blue with Dapi and the green deposits represent FITC tagged secondary antibody which was bound to the anti-involucrin primary antibody. The left panel is the comparable Phase contrast image.
  • Figure 26 represents immuno-histochemical staining for cytokeratin 10 in cultured epithelia of 4- 15 (Top), 4-150 (middle) and ⁇ -Irr (Bottom). The sections show only the nuclei are stained blue with Dapi while there were no deposits representing cytokeratin 10. The left panel is the comparable Phase contrast image.
  • Figure 27 represents immuno-histochemical staining for Filaggrin in cultured epithelia of 4- 15 (Top), 4- 150 (middle) and ⁇ -Irr (Bottom). The sections show only the nuclei are stained blue with Dapi while there were no deposits representing Filaggrin. The left panel is the comparable Phase contrast image.
  • Figure 28 consists of column charts representing non-proliferative feeder cell contamination expressed as percent of 3T3 out of total plated cells of 5000 per dish (Top) and percent of 3T3 out of total attached cells that included both 3T3 and keratinocytes (Bottom). Statistical analysis using Students t test revealed insignificant differences among the three groups.
  • a process of controlled growth arrest of feeder cells by a strategy of regulating their disintegration or life span and their ability in supporting the proliferation of human epidermal keratinocyte stem cells following a pulsed exposure to mitomycin C at permutations of concentration per se in the treating solution and the experimentally recognized dose per unit feeder cell number and the subsequent identification of an optimal feeder cell processing comparable to gamma irradiation for a speedy growth of keratinocytes resulting in the formation of a stratified epithelium that could be exploited towards grafting to treat certain ailments requiring resurfacing.
  • the instant invention relates to a process of preparing an optimal cellular substratum demonstrated through novel experimental derivation of mitomycin C titration protocol which is shown in sequential Figures 1 to 3 employing Swiss 3T3 cell line, in a way to optimize epidermal keratinocyte stem cell growth under artificial growth conditions.
  • the invention is a cost-effective process for producing a comparable growth promoting feeder layer as opposed to the standard but expensive gamma- irradiation method and can be exploited to create artificial epithelia for resurfacing diseased tissues like skin, cornea etc.
  • FIG. 1 A schematic representation of experimental derivation of doses from a given concentration of mitomycin C by cell density titrations by employing a popularly known Swiss 3T3 feeder cell line is shown in Figure 1. Varied number of cells ( ⁇ ) is treated to a 2 hrs pulse of Mitomycin C at different concentrations, the outcome of such exposures, which broadly included categories namely stimulatory, stationary, inhibitory and toxic, is correlated (Table 1, Figures 4 and 5) with the doses that are calculated from the following formula: -
  • the stimulatory influence of feeders is tested as shown in Figure 3 by replating the mitomycin C treated 3T3 feeder cells along with epidermal keratinocyte stem cells in Keratinocyte medium.
  • the stimulatory influence is ascertained by the progress of growth of keratinocytes and their ability to form maximal colony forming efficiency, to cover maximal growth area (Figures 10 to 20) during comparable culture time, and ability to form stratified epidermis (Figure 21) showing the key epidermal markers (Figure 23 to 27), while keeping a minimal feeder cell contamination (Figure 28, 29).
  • the most preferred concentration of mitomycin C is 4 ⁇ g per ml and the most preferred dose is 150 ⁇ g per million cells in terms of accomplishing not only irreversible growth arrest and also exerting maximal growth stimulatory influence on epidermal keratinocyte stem cells.
  • DMEM 10 per cent (Volume /volume) donor calf serum and 17.86 milli molar sodium bi carbonate under cell culture conditions of a constant temperature of 37°C and humidified 5 per cent carbon dioxide atmosphere and the cells were detached by trypsinization using 0.25 percent trypsin and 0.
  • a working bank was then generated from a frozen vial of master bank by sub-culturing two times until 6 th passage and cryo- banked.
  • the cultures were tested for Mycoplasma (Kumar et al 2008), doubling time, saturation density and absence of growth in methyl cellulose.
  • the sub-culture dilutions adopted throughout the banking procedure ranged from 1:6 tol : 10 while maintaining a seeding density of about 3 x 10 3 cells per cm 2 as recommended by supplier and the cultures were never allowed to grow to more than 50 per cent confluence.
  • Keratinocyte - feeder Co-culture Primary keratinocytes were obtained from Genlantis (Cat No. PH 10205A, www.genlantis.com) which were cryo-preserved at the end of first culture of epidermal cells isolated from healthy adult human skin biopsy after growing in a feeder-free and serum-free culture system. The cells were used for co-culture experiments with the growth arrested feeders as described adopting the basic Rheinwald-Green (1975) technique (Navasaria et al.1994).
  • the keratinocyte growth medium comprising: Dulbecco's modified eagle medium and Ham's F- 12 at 3: 1 ratio, 10 percent (Volume /Volume) Fetal Calf Serum and 10 ⁇ g ciprofloxacin, 5 ⁇ g of insulin, additional 110 ⁇ g L-Glutamine, One ⁇ g of dexamethasone, 24.32 ⁇ g of adenine, 20 ⁇ g of L- serine, 0.4 ⁇ g of hydrocortisone, 10 r
  • KGM keratinocyte growth medium
  • the cultures were passaged by initially treating with 0.03 grams of EDTA per 100 ml of PBS to remove feeder cells followed by detachment of keratinocytes using 0.08 grams of trypsin together with 0.01 grams of EDTA and 0.025 grams of glucose, each of which are per every 100 ml of the respective final solutions and viable cells were counted by trypan blue exclusion criterion.
  • This example describes experimental proof to show that the periodical feeder cell extinctions depended on a range of mitomycin C doses, expressed per unit cell number, which were derived arithmetically after a range of feeder cell densities are experimentally pulse exposed to various concentrations of mitomycin C.
  • Growth arrest Protocol Working bank Swiss 3T3 cells were put in culture as described in general methods and this 6 th passage output cells were seeded into T25 flasks (Nunc) containing 10 ml of medium and incubated for 5 days before treatment with mitomycin C.
  • ECNs Exposure Cell Numbers
  • the cells were exposed under the described standard cell culture conditions to a 2 hour-pulse of mitomycin C (Sigma-Aldrich, Catalogue number M4287), which was dissolved in Hanks Balanced Earl's Salts (HBES) and diluted proportionately with 10 ml of culture medium to yield a range of mitomycin C concentrations of 1 , 3, 4, 5, and 10 ⁇ g per ml; higher than 10 ⁇ g per ml were not included in the study because of their acute toxicity across all the ECNs.
  • mitomycin C Sigma-Aldrich, Catalogue number M4287
  • the dose expressed as ⁇ g of mitomycin C per million cells, which equals to pg per cell, was calculated by dividing the product of concentration in ⁇ g per ml and volume of treating solution in ml with exposure cell number in millions ( Figure 1). The significance of linearity between viable cell number for each time point and mitomycin C dose was tested by correlation coefficient. The categories of growth arrest outcome as defined on the basis of cell extinction trend were represented in a growth curve plot and analyzed by regression.
  • the ECN dependant differential cell disintegration by a given concentration could be the fallout of mitomycin C acting in a range of different doses per cell that would result from the varied exposure cell number, because notionally the total amount (weight/ volume) of mitomycin C, which is the product of concentration and volume of treating solution, is evenly shared by all exposed cells. This was tested, particularly at those effective concentrations of 3, 4, 5 & 10 ⁇ g per ml that brought about consistent growth inhibition on at least one of the ECNs.
  • the extinctions were correlated with a range of such doses that were arithmetically calculated by dividing the product of a given concentration, expressed in units of ⁇ g per ml and volume of treating solution in ml with exposure cell number in millions as depicted in Figure 1 thus the dose obtained was expressed as ⁇ g of mitomycin C per million cells, which equals to pg per cell.
  • the derived doses were arranged in ascending order from lower 19.5 to higher 549.7 pg per cell showing their relationship with the respective concentration and exposure cell density (Table 1), four distinct categories were identified amongst the total twelve tested combinations, on the basis of significant linearity of post-exposure viable cell number with time as tested by correlation coefficient. These are shown to reflect the overall growth curve status and the respective curves were plotted with regression analysis (Figure 5).
  • Stimulatory The resultant growth curve after 3 ⁇ g of mitomycin C per ml were employed on high ECN which showed consistent revival with 20th day cell number higher than the seeded cell number and a significant linear positive correlation (P ⁇ 0.01) between cell number and days of observation was assigned a final stimulatory status.
  • Inhibitory The growth curves following the use of 4, 10, 5, 10, 3 and 4 ⁇ g of mitomycin C per ml to treat medium, high, medium, medium, low and low ECNs, respectively, indicated consistent fall in cell number with significant (P ⁇ 0.01 to 0.05) negative linear correlation between change in cell number and progression of culture time and were grouped together as inhibitory.
  • Toxic The resultant growth curves after 5 and 10 ⁇ g of mitomycin C per ml, employed to treat the low ECN, showed rapid fall in cell number as visualized by the very first cell count at day 3 to be less than 10% of seeded cells and significant negative linear correlation (P ⁇ 0.01 to 0.05) between change in cell number and progression of culture time and were together assigned a final toxic status.
  • the inadequacies in mitomycin C induced growth arrest at a certain given range of concentrations could perhaps be optimized by controlling the initial exposure cell number of feeder cells or the volumes of treating solutions.
  • the significance of dose determination based on cell number could also be inferred from the recommended measures of controlling toxicity of chemotherapeutic agents in cancer patients by determining individualized dosing on the basis of recipient's lean body mass (Prado et al 2007) which is the difference between body mass and body fat, the former in turn is the product of cell number and average cell mass (Savage et al 2007).
  • exposure cell density variation strategy was to be adopted in an in vitro toxicology study design, it could possibly form a basis of calculating and predicting a compound's operational in vivo dose from the most active concentrations studied in vitro.
  • the range of combined factors of exposure cell density of feeders and concentration of mitomycin C at its constant volume may be substituted with corresponding permutations of volume and concentration of the latter, while keeping the exposure cell density to a stably safe constant, to achieve analogous dosing and regulate live feeder cell number.
  • This alternate strategy in order to set the exposure cell number to such a constant level that the cell population does not accumulate variants through successive passaging. Because cell lines like the 3T3s are known to gradually accumulate spontaneous variants with altered characteristics when grown by sub-culturing the high density confluent populations (Rubin an Xu 1989; Matthews 1993; ATCC product information sheet for CCL-92, www.atcc.org).
  • This example describes a strategy of controlling the net life-span of feeder cell growth arrest by regulating degree of cell disintegration through treatment of a constant exposure cell population with mitomycin C solutions constituted in a way to yield a range of volumes representing specific permutations of concentrations and the doses per unit cell number.
  • the permutations were calculated by dividing the product of pre-exposure cell number in millions and the chosen dose expressed as pg per cell with the concentration in ⁇ g per ml ( Figure 2). Further, a broader range of doses than those derived in example 1 was chosen for titrations to increase the scope of identifying best outcome by producing more apparent differential degrees of cell extinction. Each permutation is depicted as a pair of whole numbers in which the left hand side of the hyphen stands for concentration and the right hand side for dose.
  • the permutations analogously represent a range of volumes of treating mitomycin C solutions in which the derived volume is directly and inversely proportional to doses and concentrations, respectively and both these factors along with exposure cell number determine the total amount of mitomycin C in the final treating volume.
  • the minimum volume of treating solution to sufficiently submerge the entire cell monolayer and the maximum capacity of a chosen culture flask being the lower and upper limits, respectively, the choice of permutations to be included in the study is restricted to only a correspondingly limited range of volumes.
  • Each of the remaining intermediate concentrations of 4 and 5 ⁇ g per ml was subdivided into doses of 15, 75, 150, and 450 pg per cell and the results were compared with 3- 10 and 10-30.
  • the cells were treated under the described standard cell culture conditions with a 2 hour-pulse of mitomycin C (Sigma- Aldrich, Catalogue number M4287), which was dissolved in Hanks Balanced Earl's Salts (HBES) and diluted proportionately with culture medium to yield a desired dose permutation of mitomycin C concentrations.
  • HBES Hanks Balanced Earl's Salts
  • a median volume of culture medium containing HBES was used to expose the vehicle-control flasks which were maintained under identical culture conditions.
  • the experiments were performed to study both short-term and long term effects of mitomycin C treatments as per the following design:
  • line diagrams were constructed by plotting viable 3T3 cell number on y-axis against post- treatment time points on x-axis to show periodical cell extinctions caused by each permutation of a given mitomycin C concentration.
  • Viable cell numbers at each time point was subjected to one way ANOVA to verify the significance of variance among dose permutations of a given concentration. Additionally, the overall significance of variance across all time points among all permutations under each concentration was analyzed by two-way ANOVA; Further, linear trend lines were plotted using viable cell counts on y axis against doses on x axis by least squares fit and R 2 values were calculated by regression analysis.
  • column graphs were constructed in which each cluster of columns represented the specified permutation and each column represented viable cell numbers at the specified time points. Each cluster of 3- 10 and 10-30 was compared with every dose permutation by Students t test after pairing the corresponding time points.
  • the differential cell extinctions were also microscopically noticeable among permutations of 15, 150 or 450 pg per cell (Figure 8).
  • the vacuolated cells which are signs of cellular disintegration were rare in feeders of 4-15, less frequent in 4- 150 (C), but become apparently numerous in 4-450 (E) after 6 days post-mitomycin C exposure time point. This is further correspondingly reflected by loss of overall cellularity on culture surface after 12 days which ranged from fair in 4- 15 (B), moderate in 4- 150 (D) to very poor in 4-450 (F). Conspicuously, the cells gradually assumed broader aspect as their number depleted.
  • the post exposure cell disintegration rate was proportional to the calculated dose increments, although, the phenomenon is limited to the moderately acting intermediate concentrations and is also comparable to their similar action after cell density titrations of example 1.
  • the exhibition of such variation at lower and higher concentrations appears to relate with their weak and intense action potential, respectively, at the very first place.
  • the cell density variation strategy alone may not precisely project the effective combinations of concentrations, doses and exposure cell densities, but potentially provides primary estimates of probable range of such useful permutations for the subsequent convenient testing by corresponding volume titrations. It is thus shown that the volume titrations of a range of given concentrations, but limited to a median effective range, were instrumental in either diminishing or accentuating the net cell viability and could exert objective-specific array of consequences.
  • concentration is the term of reference for in vitro studies and the in vitro dose response curve truly represents concentration dependant evaluation, while dose is specific for in vivo studies (Eisenbranda et al 2002).
  • concentration and dose per given cell population were considered as discrete variables while making endpoint observation on cell death. Therefore, it is plausible to propose that if volume variation strategy was to be adopted following cell density titrations in an in vitro toxicology and/ or pharmacological study design, it could perhaps form the basis of extrapolating a compound's operational in vivo dose from the most active permutation of concentration and dose studied on a fixed population of cells in vitro.
  • volume titrations while using a range of less toxic intermediate concentrations of mitomycin C in regulating the viability of feeder cells unreported in prior art, it may be proposed that the approach could be superior, reliable and convenient for rendering the feeder cell growth arrest than by the cell density regulation alone. Therefore, a strategy may be adopted to test the range of feeder cell batches produced by volume titrations as demonstrated in this example in an epidermal keratinocyte co-culture model to verify if they influence differential stimulation of target cells and to subsequently identify the best outcome while comparing with the standard y-Irr feeders. The process, if proven, will overcome the reported inadequacies of the cost- effective mitomycin C approach as compared to gamma-irradiation technique.
  • the short-listed doses included for growth assessment were 15, 150 & 450 pg per cell, each combined with concentrations of 4 and 5 ⁇ g per ml, respectively.
  • Feeder groups of 3- 10 and 10-30 were included along with ⁇ -Irr feeder cells as controls for comparison.
  • Keratinocytes used in the experiments included either the frozen 1 st passage cells as received from the supplier (Genlantis) or the 3 rd passage cells produced by culturing the frozen 2 nd passage cells, both of which were subcultured using 15000 feeders of 4-150 group per cm 2 .
  • CFE Colony forming Efficiency
  • digital image analysis for growth area assessment were performed plating low density keratinocytes over various feeder groups in 6-well plates with each well containing 15,000 feeder cells per cm 2 .
  • CFE was performed using 250 keratinocytes of 3 rd passage and feeders treated with all the short-listed permutations.
  • separate experiments were performed to estimate average colony size and total growth area with 170 and 340 viable 1 st passage keratinocytes plated per well, respectively, and the feeders were the best performing 4- 150 along with the sub-optimal 4- 15 and ⁇ - ⁇ .
  • CFE comprised of counting of discrete keratinocyte colonies of 8 or more cells, expressed as percentage of plated cells. Estimation of average colony size was based on growth area assessment and CFE, wherein small colonies of highly irregular shape containing broad, flattened and terminally differentiated cells were considered as aborted and the rest constituted the proliferative colonies. Experiments for CFE were performed in triplicate while growth area assessment was performed in quadruplicate per feeder group.
  • the images were triplicated, out of which two were used for isolating the two colors, while the third un-treated one was referred for comparison. Once the respective colors were isolated, they were superimposed to verify the reproduction of an image similar to the original. This was followed by attainment of quantification, in terms of number of pixels within the selected red area, using the Histogram command in the Image menu. The percentage of number of red pixels out of total number of pixels of the well was calculated and area of keratinocyte growth was derived from the known total area of the well. In case of wells plated with 170 keratinocytes, the calculated area in square centimeters was divided by the total number of colonies counted in the respective well to obtain average colony size.
  • BrdU labelling was estimated in slide flasks (Nunc) in which cultures were initiated with 70 viable 1 st passage epidermal keratinocytes per flask containing the best performing feeders of 4- 150 along with 4-15 and D-Irr at a density of 15,000 per cm 2 and incubated with change of culture medium every alternate day for 10 days.
  • Feeder cells were selectively removed with 0.02 % EDTA and the wells were incubated in non radio-active Bromodeoxy Uridine (BrdU) for 1 hour, fixed in Cornoy's fixative, treated with 4M HC1 for antigen retrieval, neutralized with 0.1M sodium Borate, incubated in primary mouse monoclonal anti-BrdU antibody (Cat No.sc-32323, Santacruz) followed by visualization of fluorescence labelled nuclei after incubating in FITC labeled anti-mouse secondary goat polyclonal antibodies (Cat No. sc- 2010, Santacruz Biotechnology Inc.). Every colony from triplicated slide flasks per feeder group was differentially counted for labelled and unlabeled nuclei.
  • PrdU non radio-active Bromodeoxy Uridine
  • each colony was photographed in both phase contrast and fluorescence modes on Nikon Diphot 300 microscope at 20X objective using Evolution QEi monochrome camera (Media Cybernetics); multiple images were taken if the colony was larger than the field and overlapping margins were demarcated by matching the images before counting of nuclei was performed using manual tag tool of Image Pro-Express express software version 6.0.
  • Co-cultures were initiated in 24 well plates using 3 rd passage primary epidermal keratinocytes, in the presence of short-listed feeder groups with change of culture medium every alternate day until differential cell counts performed from triplicate wells per group at days 3, 6 & 9.
  • the cultures were first treated with 0.02 % EDTA to selectively remove the feeder cells that were collected in keratinocyte medium followed by collection of keratinocytes in separate vials after detaching them using a 3 times diluted solution of 0.25 % trypsin, 0.03 per cent EDTA and 0.025 per cent glucose. Viable cell counts were performed in Neubauer chamber after trypan blue exclusion.
  • the artificial epithelia which are equivalent of cultured epithelial autografts were prepared using the best performing feeder cells of 4- 150 and the other sub-optimally performing feeders of 4- 15 along with ⁇ - ⁇ were included for comparison.
  • Triplicate cultures were initiated in 6-well plates by seeding 800 viable 1 st passage keratinocyte cells per cm 2 and 15,000 feeder cells per cm 2 .
  • Culture medium was changed every alternate day until confluence and Epidermal growth factor (EGF) was added to the culture medium after 48 hours.
  • EGF Epidermal growth factor
  • Two wells were used for histological and immunohistochemical studies, while the third was assessed for feeder cell contamination and growth in agar-methyl cellulose.
  • the stratified epithelium from confluent keratinocyte cultures was recovered by incubating in solution containing 2 mg of Dispase per ml of keratinocyte culture medium without serum at 37°C for 50-70 minutes.
  • Histological evaluation The epithelia were fixed with 4% paraformaldehyde and processed for paraffin embedding by dehydrating through graded series of alcohol followed by clearing in xylene and embedding in paraffin. 5 pm thick sections of cultured epidermis were deparaffmized, hydrated, stained with Haematoxylin and Eosin and mounted in DPX after dehydrating.
  • Immunohistochemistry Parallel sections were deparaffmized in xylene and hydrated through graded series of ethanol. Antigens of filaggrin, cytokeratin- 10 (CK- 10) and cytokeratin- 14 (CK- 14) were retrieved by immersing the slides containing sections in lOmM of sodium citrate buffer for 30 minutes at 90°C followed by cooling for 30 minutes, while involucrin was retrieved by treating for 7 min at room temperature with freshly prepared 0.1% trypsin and 0.1% CaC in Tris Buffered Saline (TBS).
  • TBS Tris Buffered Saline
  • Sections were subsequently washed with TBS, blocked with 2 % of normal goat serum (SC-2043) for 1 hr at 37°C in a humidified chamber and incubated overnight at 4°C in mouse monoclonal primary antibodies (Santacruz Biotech) against filaggrin (SC-25896), Involucrin (SC-21748), CK- 10 (SC-51581), CK- 14 (SC-58724) diluted at a ratio of 1 :50. Sections were then rinsed with TBS before incubating for 1 hr at 37°C in FITC- tagged goat anti-mouse IgG secondary antibody (SC-2010) diluted at 1 : 100 followed by mounting in DAPI containing medium (SC-24941). Negative controls were equivalently processed except for incubating in plain TBS in place of primary antibody. Normal human skin processed similarly served as positive control.
  • the third confluent keratinocyte culture was disaggregated using a 3 times diluted solution containing 0.25 % trypsin, 0.03 per cent EDTA and 0.025 per cent glucose and the cell suspension was used for assessing the feeder cell contamination and growth of keratinocytes in agar methyl cellulose.
  • Feeder contamination The isolated keratinocyte suspension from the 3 feeder groups was assessed for both (a) non-proliferative and (b) proliferative feeder cell contamination by Hoechst staining,
  • Non-proliferative contamination The cell suspension generated by the three feeder groups was plated in 60 millimeter dishes without feeders at a density of 5000 cells per dish; cells were allowed to attach overnight by incubating in KGM and number of feeder cells were counted after Hoechst staining.
  • Proliferative contamination The trypsinized cells were plated in T25 flasks at a density of 50,000 cells per flask and incubated in KGM for a period of 3 weeks and processed for Hoechst staining.
  • Hoechst staining Cultures at the end of respective incubation periods were fixed in a chilled solution of three parts of methanol and one part of glacial acetic acid, stained with Hoechst 33258 (Sigma, H-6024) at a concentration of 0.125 ⁇ g per ml of Hank's balanced salt solution, in the dark for 10 minutes. The dishes were washed with distilled water and observed in a fluorescence microscope (Nikon Diaphot 300) fitted with excitation filter of 330-380 nm and emission filter at 420 nm. 3T3 cells were distinguished from Keratinocytes on the basis of their different nuclear size, morphology and fluorescence pattern (Alitalo et al., 1982).
  • Transformation assay 5 X 10 4 isolated cells from trypsinized confluent keratinocyte cultures grown in presence of the three feeder groups were suspended in Methyl cellulose (Methocel, Sigma-Aldrich). Methyl cellulose was prepared at a final concentration of 0.8% in 3T3-CBS medium, poured over a base of 0.6% agar in 35 millimeter dish, incubated at standard culture conditions and examined under Nikon inverted phase contrast microscope after 2 weeks to look for keratinocyte growth.
  • Methyl cellulose Methyl cellulose
  • the calculated critical values for the Tukey HSD test were used for post-hoc analysis to estimate the real significant difference between comparable time points of two feeder groups.
  • 4- 150 produced highly significant (P ⁇ 0.001) stimulation than 5- 150; hence subsequent experiments on growth area included appraisal of 4- 150 in comparison with 4- 15 and ⁇ -Irr, which are considered as standard feeders.
  • the percent of labeled cell number in ⁇ -Irr was found to be 28.9% (3270/ 1 1321) from 21 colonies and was not significantly different from that in 4- 150, but at the same time, ⁇ -Irr was found to be significantly superior to 4- 15.
  • feeders of 4- 150 were superior to 4- 15 in inducing a higher BrdU label, in spite of absence of significant variation in average cellularity per colony, indicating that an overall high cell turn over would eventually set in.
  • the importance of dose titration of feeders with mitomycin C is further highlighted through improvement of the otherwise sub-optimal performance to match with that of ⁇ -Irr feeders in achieving faster growth of keratinocytes.
  • the cell death in ⁇ -Irr feeders was significant and the most rapid compared to any of the MMC feeders.
  • Stratified epithelium Generally, Keratinocyte colonies of 2 to 5 cells appeared in the well by 2 nd day in cultures. After one week, large keratinocyte colonies with specific areas of differentiation appeared. Keratinocyte colonies eventually coalesced and the confluent cultures gave rise to stratified squamous epithelium in 10- 13 days which were isolated from culture surface by dispase treatment. The isolated epithelial sheets contracted to about 3 ⁇ 4 the size of the well, appeared as thin, fragile, semi- translucent, free-floating tissues (Figure 21).
  • Histological characterization The H & E stained paraffin sections revealed that the epidermal constructs grown over feeders of 4-150 uniformly presented the basal and the differentiated compartments with more or less even stratification, where as the epidermal thickness resulting from its growth in presence of either ⁇ -Irr or 4-15 MMC feeders was highly varied from one cell to 8 cells ( Figure 22). Particularly, the sheets from ⁇ -Irr group were very inconsistent with intermittent regions of completely differentiated segments in between zones of good thickness showing well formed basal compartment.
  • Non-proliferative contamination The disaggregated cultured epithelia grown in presence of feeders of 4- 15, 4- 150 and ⁇ -Irr revealed 0.46 ⁇ 0.03, 0.54 ⁇ 0.04 and 0.56 ⁇ 0.09, respectively as percentages of attached 3T3 cells out of 5 X 10 3 plated cells ( Figure 28), which worked out to be 3.3 ⁇ 0.2, 3.5 ⁇ 0.3 and 3.6 ⁇ 0.6, respectively, as percentages of feeder contaminants out of the total cells attached and the rest were keratinocytes. The data showed no significant differences.

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Abstract

La présente invention concerne un système de culture utilisable en vue de la multiplication de cellules souches des kératinocytes épidermiques, ledit système consistant : a. à traiter les cellules nourricières de façon peu coûteuse à l'aide de mitomycine c ; b. à utiliser un substrat se présentant sous la forme de ces cellules nourricières ; et c. à obtenir une meilleure stimulation de la multiplication des cellules souches des kératinocytes épidermiques qu'avec des cellules nourricières exposées à des rayons γ. Ce système est caractérisé en ce que la concentration optimale en mitomycine c est encore combinée avec la dose par cellule pour obtenir une plage de permutations concentration-dose, où la concentration en mitomycine c se situe dans un intervalle de 3 à 10 µg par ml et la dose dans une plage de 15 à 450 pg par cellule.
PCT/IN2015/000404 2014-10-30 2015-10-30 Procédé de traitement de cellules nourricières adaptées à la prolifération de cellules souches adultes WO2016067306A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001005942A2 (fr) * 1999-07-20 2001-01-25 Epitech S.A. Culture amelioree et utilisations de keratinocytes
WO2006101834A1 (fr) * 2005-03-17 2006-09-28 Stratatech Corporation Substituts de la peau a purete amelioree

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001005942A2 (fr) * 1999-07-20 2001-01-25 Epitech S.A. Culture amelioree et utilisations de keratinocytes
WO2006101834A1 (fr) * 2005-03-17 2006-09-28 Stratatech Corporation Substituts de la peau a purete amelioree

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NIETO ET AL: "Effect of mitomycin-C on human foreskin fibroblasts used as feeders in human embryonic stem cells: Immunocytochemistry MIB1 score and DNA ploidy and apoptosis evaluated by flow cytometry", CELL BIOLOGY INTERNATIONAL, ACADEMIC PRESS, GB, vol. 31, no. 3, 21 February 2007 (2007-02-21), pages 269 - 278, XP005894320, ISSN: 1065-6995, DOI: 10.1016/J.CELLBI.2006.11.006 *
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