Field of the Invention:
The present invention is in the field of cell biology and more particularly to a process of proliferation of keratinocyte cells using a novel culture system.
Background of the Invention:
Keratinocyte is the major constituent of the epidermis, constituting 95% of the cells found in the epidermis. Cultivated keratinocytes replicate readily and can generate large numbers of cells which exhibit certain features of squamous differentiation in vivo. Hence such cultivated cells can be used in several manners such as wound healing, skin grafting etc. For instance, autologous keratinocytes obtained from small skin biopsy specimen from burned patients and cultured in vitro are used to treat severely burned patients.
However, epidermal keratinocyte stem cells and certain embryonic stem cells do not readily grow in simple culture media and such cells depend on a feeder layer consisting usually of fibroblast cells like 3T3 mouse embryonic skin fibroblast cells that are attenuated (growth arrested). Attenuated feeder layer is produced by exposing feeder cells to gamma-irradiation or to cell proliferation-blocking chemical agents like Mitomycin C so that they do not undergo cell division but remain metabolically functional in a way to impart growth stimulatory influence on target cells.
The concentration of the Mitomycin C used is critical; too low a concentration would result in improper attenuation, resulting in the reversal of attenuation, which would in turn result in growth of the attenuated cells contaminating the target cells. On the other hand, too high a concentration would result in total destruction of the cell rendering it useless as a feeder layer. In prior art, several concentrations of Mitomycin C has been used. However, the concentrations have either been high or low; consequently an effective attenuated layer has not been produced.
Hence, there is unmet need to develop a culture system in which the mitomycin C induced attenuation is irreversible while maintaining effective feeder capability.
Objects of the Invention:
An object of the present invention is to provide a culture system in which the mitomycin C induced attenuation is irreversible.
Statement of the Invention:
A culture system for the growth of epidermal keratinocyte stem cells comprising of inactivating the feeder cells effectively with Mitomycin C; and providing a substrate in the form of such feeder cells; achieving effective feeder cell growth arrest in terms of permanent loss of growth in them while maximally stimulating epidermal keratinocyte stem cell growth; characterized in that the concentration of mitomycin C is titrated with respect to the number of cells.
Summary of the Invention:
The present invention relates to the culturing of human epidermal keratinocyte stem cells using growth arrested cells as feeder layer, such that the attenuation achieved is irreversible and the stimulation of keratinocyte cell proliferation is maximum. This is achieved by using a concentration of mitomycin C that is titrated with respect to the number of cells.
Brief Description of the Drawings:
Figure 1 shows the growth curves and the corresponding linear trend lines representing cell counts over a 20-days period in cultures initiated by re-plating 3T3 cells that were previously exposed to 5 micrograms Mitomycin C per milliliter at doses of 5, 10, 15 and 20 micrograms per million cells for a 2-hours pulse. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis.
Figure 2a shows discrete colonies (encircled) of bipolar 3T3 cells that appeared after 8 days in culture subsequent to exposure to 5 micrograms Mitomycin C per milliliter at a dose of 5 micrograms per million cells for a 2-hour pulse. The colonies show signs of
merging (broad arrow). The attenuated cells are well spread (solid arrow). (Phase-contrast 100 X)
Figure 2b shows a growing colony of 3T3 arising from those cells reverted to proliferation after exposure to 5 micrograms Mitomycin C per milliliter at a dose of 5 micrograms per million cells for a 2-hour pulse. (Phase-contrast 200 X)
Figure 2c shows a large expanding colony of proliferative 3T3 cells showing migratory patterns after exposure to 5 micrograms Mitomycin C per milliliter at a dose of 5 micrograms per million cells for a 2-hour pulse. The attenuated cells are well spread out (arrow) as seen in the background. (Phase-contrast 100 X)
Figure 3 shows a growth curves representing cell number of 3T3 and keratinocytes (HEK) on days 3, 6 and 9 after they were plated together. The broken lines are of 3T3 which were attenuated with 1 micrograms Mitomycin C per milliliter at a dose of 10 µg/106 cells and 5 micrograms Mitomycin C per ml at doses of 5, 10, 15 and 20 micrograms per million cells for a 2-hours pulse as represented by 1—10, 5—5, 5—10, 5— 15 and 5—20 and 0—0 representing untreated 3T3 cells, while the bold lines represent keratinocytes that were plated with the respective 3T3. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis.
Figure 4 shows a culture of keratinocytes at 4th passage 2 hours after they were plated without attenuated 3T3 feeders showing 3T3 cells as contaminants (solid arrows) recovered and accumulated from the preceding passages that were propagated employing 3T3 attenuated with 5 micrograms of Mitomycin C per milliliter at a dose of 10 micrograms per million cells. Well-adhered keratinocytes (open arrow) are seen amongst several poorly anchored ones while 3T3 are bipolar and adhered. (Phase-contrast 400 X)
Figure 5a shows an aborted colony of keratinocyte cells surrounded by proliferating and migratory 3T3 cells attenuated with 5 micrograms Mitomycin C per milliliter at a dose of 10 micrograms per million cells in an 8 days old 6th passage keratinocyte culture.
Disintegrating 3T3 cells (arrows) are visible over the proliferating 3T3 cells. (Phase-contrast 125 X)
Figure 5b shows a keratinocyte colony (Kc) and the clumps of disintegrated 3T3 cells (arrow) are visible amongst the proliferating and migratory 3T3 cells attenuated with 5 micrograms Mitomycin C per milliliter at a dose of 10 micrograms milliliter million cells in an 8 days old 6th passage keratinocyte culture. (Phase-contrast 125 X)
Figure 6 shows the line diagram plotted with concentration-dose combinations of 1 micrograms Mitomycin C per milliliter at a dose of 10 micrograms per million 3T3 cells, 5micrograms Mitomycin C per milliliter at doses of 5, 10, 15 and 20 micrograms per million 3T3 cells for a two-hour pulse as represented by 1—10, 5—5, 5—10, 5—15 and 5-20 Mitomycin C and 0—0 representing untreated 3T3 cells and the corresponding keratinocytes grown together on x-axis and cell numbers representing 3T3 (3T3, broken lines) and keratinocytes (HEK, solid lines) on days 3, 6 and 9 (D3, D6 and D9), respectively, on y-axis, showing periodical change in cell number. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis.
Figure 7 is a scatter plot constructed with concentration-dose combinations of 1 micrograms Mitomycin C per milliliter at a dose of 10 micrograms per million 3T3 cells, 5 micrograms Mitomycin C per milliliter at doses of 5, 10, 15 and 20 micrograms per million 3T3 cells for a two-hour pulse as represented by 1-10, 5-5, 5-10, 5-15 and 5-20 Mitomycin C and 0-0 representing untreated 3T3 cells and the corresponding keratinocytes grown together on x-axis and cell numbers on y-axis with linear trend lines representing 3T3 (broken line) and keratinocytes (solid line), respectively, plotted by calculating the least squares fit through cell count data points using regression analysis. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis.
Figure 8 shows growth curves representing cell counts over a 9-days period in cultures initiated by re-plating 3T3 cells that were previously exposed to 6, 7, 8 micrograms Mitomycin C per milliliter each at doses of 10, 20 and 30 micrograms per million cells, respectively, for a two-hour pulse. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis.
Figure 9a shows keratinocyte colony in a 6-days old culture with narrow cell bodies (encircled) grown by using 3T3 feeder cells, which were growth arrested by treating with 5 micrograms Mitomycin C per milliliter at 20 micrograms per million cells. The broadly spread out feeders (arrow) are seen well-attached obstructing the spread and free migration of keratinocytes. (Phase-contrast 250 X)
Figure 9B shows keratinocyte colony (Kc) in a 6-days old culture with broadly spread out cell bodies grown by using 3T3 feeder cells, which were growth arrested by treating with 5 micrograms Mitomycin C per milliliter at 60 micrograms per million cells. The feeders are seen either loosely anchored (broad arrow) or vacuolated and degenerating (solid arrow) leaving empty spaces (encircled) and allowing free spread of keratinocytes. (Phase-contrast 250 X)
Figure 10 is a line diagram plotted with concentration-dose combinations of 5 and 6 micrograms Mitomycin C per milliliter each divided into doses of 20, 40 and 60 micrograms per million 3T3 cells, respectively and 7 micrograms Mitomycin C per milliliter at a dose of 20 micrograms per million 3T3 cells for a 2-hours pulse as represented by 5-20, 5-40, 5-60, 6-20, 6-40, 6-60 and 7-20 Mitomycin C on x-axis and cell numbers representing 3T3 cells (3T3, hollow circles connected by broken lines) and keratinocytes (HEK, solid triangles connected by solid lines) on days 3, 6, 9 and 12 (D3, D6, D9, DI2), respectively, on y-axis, showing periodical changes in cell number. The lines were interrupted to group together all the dose-dependant differential cell counts for each given day. The dimly shaded circles and triangles shown against 0-0 for 3, 6 and 9 days represent the number of untreated 3T3 cells and the keratinocytes grown together, respectively and are connected to their corresponding normally shaded markers by lines
of lighter shades. These were not shown for Day 12, since the untreated 3T3 cells reached super-confluence beyond 9 Days and both the cell types were consequently lost before 12th day during change of medium. Polynomial trend lines to the order of 2 representing 3T3 cell number (3T3, broken bold line) and keratinocytes (HEK, solid bold line), respectively, were plotted by calculating the least squares fit through cell number data points using regression analysis. Each point is the mean of cell counts from 3 wells and standard deviation is shown as error bars on y-axis.
Figure 11 is a bar diagram showing disintegration of 3T3 cells that were attenuated with Mitomycin C at concentration-dose combinations of 5 and 6 micrograms Mitomycin C per milliliter at doses of 20, 40 and 60 micrograms per million 3T3 cells and 7 micrograms Mitomycin C per milliliter at a dose of 20 micrograms per million 3T3 cells for a 2-hours pulse represented by 5-20, 5-40, 5-60, 6-20, 6-40, 6-60 and 7-20 Mitomycin C as plotted on x-axis. The extent of cell density as plotted on y-axis was recorded by three independent observers who assessed weekly progress until 4 weeks by assigning grades between five and zero, representing highest density as on day-one and no attached cells on plastic surface, respectively. (A) Bar diagram representing data from 24-well plates wherein each well is inoculated with attenuated 3T3 cells at the density of 25000 cells per square centimeter. (B) Bar diagram representing data from 75 square centimeter culture flasks inoculated with attenuated 3T3 cells at the density of 35,000 cells per square centimeter.
Figure 12 shows disintegration of 3T3 cells exposed for a two-hour pulse Mitomycin C 2 weeks following reseeding in culture flasks. (A) Moderate disintegration of 3T3 cells exposed to 5 micrograms Mitomycin C per milliliter at a dose of 20 micrograms per million cells which is represented by numerical dose of 5-20,. Well spread out cells with high cytoplasm to nucleus ratio (arrows) are frequent. (B) Higher level of disintegration of 3T3 cells exposed to 5 micrograms Mitomycin C per milliliter at a dose of 60 micrograms per million cells which is represented by numerical dose of 5-60. Majority of the cells are disintegrated or vacuolated and those with intact nuclei are rare. (Phase-contrast 200 X)
Figure 13 shows the appearance of morphologically distinct clones (a, b and c) in a 5-weeks old culture of an inadequately growth arrested 3T3 feeder cells. The inadequate attenuation was produced by exposing 3T3 cells for a two-hour pulse to 30 milliliters of a solution containing 5 micrograms Mitomycin C per milliliter at a density of 200,000 cells per every square centimeter in a 75 square centimeter culture flask corresponding to 10 micrograms Mitomycin C per million feeder cells and representing a numerical dose of 5-10. The treated 3T3 cells were re-plated in keratinocyte growth medium to simulate feeder based keratinocyte culture system at a density of 35,000 cells per square centimeter in a 75 square centimeter. (Phase-contrast 125 X)
Detailed Description of the Invention:
Accordingly, the present invention provides a culture system for maintaining the growth of epidermal keratinocyte stem cells comprising:
a. inactivating the feeder cells effectively with Mitomycin C; and
b. providing a substrate in the form of such feeder cells;
c. achieving effective feeder cell growth arrest in terms of permanent loss of growth in
them while maximally stimulating epidermal keratinocyte stem cell growth;
In particular, the present invention pertains to using a concentration of mitomycin C, such that the concentration is adjusted in terms of the number of cells, i.e. the concentration of mitomycin C is further adjusted per million feeder cells.
For the purpose of the present invention a culture system for the growth of epidermal keratinocyte stem cells comprises of a cellular substrate in the form of growth arrested feeder cells. The feeder cells can be any cell, but are preferentially murine embryonic dermal fibroblast 3T3 cells. Similarly, this culture system could be advantageously used for the growth of other cells such as embryonic stem cells.
It is further submitted that the cells can be obtained from any public resource and not necessarily from foetus.
The growth arrest of 3T3 mouse fibroblast cells is produced by treating with a precise treatment volume of Mitomycin C solution of a given concentration of Mitomycin C to prevent their multiplication in an irreversible manner and to produce maximal growth stimulation of epidermal keratinocytes. The growth arrested 3T3 feeder cells are prepared by the steps of:
a. growing the feeder cells in 3T3 medium comprising of Dulbecco's modified eagle
medium supplemented with 10% donor calf serum, 10 micrograms ciprofloxacin
per milliliter to a density of 200,000 cells every square centimeter in 75 square
centimeter culture flask which is equal to 15 million cells per flask;
b. calculating the specific volume of treating solution of Mitomycin C arithmetically
from its given concentration and the requisite quantity of Mitomycin C to be made
available per million feeder cells in order to produce cell number dependant
differential growth arrest;
c. illustration of numerical doses of Mitomycin C comprising of two numbers
separated by a dash, the first number of which represents the concentration of
Mitomycin C as micrograms per milliliter followed by a second number denoting
dose of Mitomycin C in micrograms per million feeder cells;
d. exposing 15 million feeder cells for a one to three hour pulse, preferentially, two
hour pulse to the following varied volumes of mitomycin C solution prepared in
3T3 medium;
i. exposing the 15 million 3T3 feeder cells to 15, 30, 45, 60, 120 and 180 milliliters of Mitomycin C solution having a concentration of 5 micrograms per milliliter which correspond to the doses of 5, 10, 15, 20, 40 and 60 micrograms Mitomycin C per million cells, respectively and are denoted by numerical doses of 5-5, 5-10, 5-15, 5-20, 5-40 and 5-60 respectively;
ii. exposing 15 million 3T3 feeder cells to 25, 50, 75, 100 and 150 milliliters of Mitomycin C solution having a concentration of 6 micrograms per milliliter which correspond to the doses of 10, 20, 30, 40 and 60 micrograms Mitomycin C per million cells, respectively and are denoted by numerical doses of 6-10, 6-20, 6-30, 6-40 and 6-60, respectively;
iii. exposing 15 million 3T3 feeder cells to 21.429, 42.857 and 64.286 milliliters of Mitomycin C solution having a concentration of 7 micrograms per milliliter which correspond to the doses of 10, 20 an 30 micrograms Mitomycin C per million cells, respectively and are denoted by a numerical doses of 7-10, 7-20 and 7-30;
iv. exposing 15 million 3T3 feeder cells to 18.75, 37.5 and 56.25 milliliters of Mitomycin C solution having a concentration of 8 micrograms per milliliter correspond to the doses of 10, 20 an 30 micrograms Mitomycin C per million cells, respectively and are denoted by a numerical doses of 8-10, 8-20 and 8-30;
e. replating the mitomycin C treated 3T3 feeder cells either alone or as co-culture with epidermal keratinocyte stem cells in Keratinocyte 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 micrograms ciprofloxacin, 5 micrograms of insulin, additional 110 micrograms L-Glutamine, One microgram of dexamethasone , 24.32 micrograms of adenine, 20 micrograms of L-serine, 0.4 micrograms of hydrocortisone, 10 nanograms of Choleratoxin, 10 nanograms of Epidermal Growth Factor, 1.346 nanograms of Tri-Iodo-thryronine and 5 micrograms of Transferrin, each of which are per every milliliter of growth medium.
The mitomycin C concentrations used to inactivate the growth of feeder cells are in the range of 5 to 8 micrograms per milliliter while the doses are in the range of 5 to 60
micrograms per million cells. All the numerical doses beginning with a concentration of 7 and 8 micrograms per milliliter are lethal and such a quick loss of growth-arrested feeder cells is highly disadvantageous to the growth of keratinocyte stem cells.
The most preferred concentration of mitomycin C is 5 micrograms per milliliter and the most preferred dose being 60 micrograms per million cells. Thus, the most preferred least numerical dose is 5-60 in terms of accomplishing irreversible growth arrest and also exerting maximal growth stimulatory influence on epidermal keratinocyte stem cells. This numerical dose is ideal for maintaining a pure epidermal keratinocyte culture as tested up to 6 passages without any contamination by 3T3 feeder cells through curtailment of attenuation reversal. The other numerical doses of 5-5 and 5-10 falling within the same concentration of 5 micrograms per milliliter produced deficient attenuation as evidenced by reversal of growth arrest pointing that numerical dosing is a very practically useful procedure. Such a deficient attenuation when employed in the co-culture system resulted in revelation of contamination of epidermal keratinocyte cultures with the reverted 3T3 cells after 4 to 6 sub-cultures. Furthermore, reseeding such deficiently attenuated 3T3 feeders without keratinocytes resulted in the formation of morphologically distinct and proliferative clones of 3T3 cells.
The present invention demonstrates that the deciding factors for volume fixation are the exposure cell number, concentration and dose per cell number and reproducibility of results is attained by invariably keeping the exposure cell number constant among all flasks treated in a single batch.
The invention is illustrated by the following examples which are only meant to illustrate the invention and not act as limitations. All embodiments apparent to a process there in the art are deemed to fall within the scope of the present invention.
EXAMPLES
General protocols
Cell culture: Murine embryonic dermal fibroblast NIH 3T3 cells (obtained from the National Center for Cell Science, Pune, India) were grown in 3T3 growth medium comprising of Dulbecco's modified Eagle's medium (DMEM, GIBCO, 12800-058) supplemented with 10 per cent (Volume /Volume) donor calf serum, Hyclone SH 30073.03), ciprofloxacin (10 micrograms per milliliter, Indian Pharmacopia) in a humidified 5 per cent carbon dioxide atmosphere at 37 degrees centrigrade. Primary Keratinocytes (Genlantis, USA) were grown in keratinocyte growth medium (KCM) which comprised of the following ingredients:
i. Dulbecco's modified eagle medium (GIBCO, 12800-058) and Ham's F-12
(GIBCO, 21700-026) at 3:1 ratio
ii. 10 percent (Volume /Volume) Fetal Calf Serum, (Hyclone, SH30071.03)
iii. 10 micro grams ciprofloxacin per milliliter (Indian Pharmacopia)
iv. 5 micrograms of insulin (human recombinant, Sigma I 2767) per milliliter,
v. additional 110 micrograms L-Glutamine (Sigma, G3126) per milliliter
vi. One microgram of dexamethasone (Indian Pharmacopia) per milliliter
vii. 24.32 micrograms of adenine (Sigma, A 8626) per milliliter
viii. 20 micrograms of L-serine ( Sigma, S4311) per milliliter
ix. 0.4 micrograms of hydrocortisone (Sigma, H3160) per milliliter
x. 10 nanograms of Choleratoxin (Sigma, C9903) per milliliter
xi. 10 nanograms of Epidermal Growth Factor (In Vitrogen, 13247-051)
xii. 1.346 nanograms of Tri-Iodo-thryronine per milliliter(Sigma, T6397)
xiii. 5 micrograms of Transferrin (Sigma T8158) per milliliter
Standard Mitomycin C treatment Protocol: Exponentially growing NIH 3T3 fibroblasts (NCCS, Pune, India) were seeded into 75 square centimeter-sized sterile plastic culture flasks (Easy Flask, Nunc) at a density of 15000 cells per square centimeter and allowed to grow for 6 days until a density of about 200,000 cells per square
centimeter was reached as confirmed by cell counts in at least 3 parallel flasks before exposure to Mitomycin C (Sigma). Cells were exposed to various concentration (micrograms per milliliter) and dose (micrograms per million cells) combinations of Mitomycin C prepared in 3T3 medium from a stock of 200 micrograms per milliliter of HEPES's Buffered Earl's Salts (HBES), for a two-hour pulse. Each concentration-dose combination is expressed as a unique numerical dose comprising of two numbers separated by a dash, the first number denoting concentration of Mitomycin C as micrograms per milliliter followed by second one denoting dose of Mitomycin C as micrograms per million feeder cells. The requisite doses were attained by appropriately adjusting the volume of culture medium containing a given concentration of Mitomycin C. Subsequently, the cells were washed thrice in 3T3 medium with three changes every 15 minutes, detached with 0.1 per cent of trypsin and 0.2 per cent of Ethylene Diamine Tetra Acetic Acid (EDTA) solution and viable cells were estimated based on cell counts using Neubauer chamber after trypan blue exclusion. The cell number obtained after treatment with Mitomycin C was compared with that obtained from untreated parallel flasks. The treated cells were frozen in liquid nitrogen at a density of 12 million cells per milliliter of 3T3 medium containing 10 per cent donor calf serum and 10 per cent dimethyl sulfoxide (volume/volume) until 24 hours when they were re-plated alone to check their growth arrest and/or as co-culture with human epidermal keratinocyte stem cells to check growth stimulatory influence on the latter. For re-plating, the attenuated 3T3 cells were quickly thawed, washed thrice in 3T3 medium, viable cell counts were performed and the requisite number of cells was suspended in KCM for seeding with or without keratinocytes in cultures. The culture medium was changed every alternate day. All the experiments were performed under sterile conditions in triplicate and repeated at least twice.
Example 1:
Attenuation failure depends on dose per cell:
In an experiment designed to verify dependency of attenuation on the number of cells present at the time of exposure to Mitomycin C, a fixed number of 3T3 cells was exposed to varying volumes of Mitomycin C solution in 3T3 culture medium prepared at a
concentration of 5 micrograms per milliliter. The 3T3 cells were grown in 75 square centimeter flasks to reach a density of 200,000 cells per every square centimeter which is 15 million cells per flask. One flask each was exposed for 2 hours to 15, 30, 45 and 60 milliliters of Mitomycin C solution corresponding to the chosen doses of 5, 10, 15 and 20 micrograms Mitomycin C per million cells, respectively and are denoted by numerical doses of 5-5, 5-10, 5-15 and 5-20, respectively. The rest of the procedure was as per the standard Mitomycin C treatment protocol described above. The freshly treated cells were re-plated in triplicate wells of 24 well plates at a density of 10,000 cells per square centimeter (20,000 cells per well) and viable cell counts were undertaken by Trypan blue exclusion method every alternate day until 10 days followed by a final count on day 20.
Statistics: Growth curves with linear trend lines were plotted by calculating the least squares fit through data points using regression analysis. The influence of various concentration-dose combinations on cell numbers of 3T3 was statistically analyzed by Student's t-Test.
Results and Discussion: The growth curves (Figure 1) representing cell numbers after exposure to Mitomycin C at numerical doses of 5-5 and 5-10 showed significant (P<.001) increase in cell number on day 8 and 20, respectively, over 5-15 and 5-20, which showed more or less unchanged cell number throughout the observed period. The increase in cell number was apparently caused by resumption of proliferative activity as observed in few foci among the largely well spread out and disintegrating cells. The cells in such proliferative foci were mostly bipolar and formed discrete colonies (Figure 2A) that gradually grew larger (Figure 2B) and showed migratory patterns (Figure 2C). The cell number increase following the numerical dose of 5-5 was more pronounced by day 20 as compared to that of 5-10, since failure of growth arrest in the former appeared earlier than in the latter. Correspondingly, the linear trend lines representing numerical doses of 5-5, 5-10, 5-15 and 5-20 revealed R2 values of 0.75, 0.39, 0.07 and 0.05, respectively.
The results evidently demonstrate that the attenuation protocol employing numerical
doses compromising of varied doses expressed as micrograms per million cells but within the same concentration of 5 micrograms per milliliter brings about differential extent of attenuation in terms of a more detrimental reversal of growth-arrest. This inadequate attenuation is more damaging which inadvertently would end up in the resumption of feeder cell proliferation and consequently contaminating the target cells. The differential growth arrest, which is based on cell density-dependent numerical dosing, could be the main causative factor for the reported contradictions on efficacy of Mitomycin C (Schrader 1999; Roy et al., 2001), since our unique procedural approach is based on an earlier proposition by one of us (Yemeni and Jayaraman 2003) and has never been considered and adopted by any investigator to date.
Example 2:
Growth support by differentially attenuated 3T3:
In order to estimate the ability of differentially growth arrested 3T3 cells in supporting the growth of target stem cells, experiments were conducted by seeding primary human epidermal keratinocytes (Genlantis) into 24 well plates and growth arrested 3T3 cells at densities of 5000 (10,000 per well) and 15000 cells (30,000 per well) per square centimeter, respectively. The cells were grown in kertinocyte growth medium with a change every alternate day. The attenuation protocol of 3T3 was as per example 1. In brief, 3T3 cells at a density of 200,000 per square centimeter were exposed for 2 hours to culture medium containing a final concentration of 5 micrograms Mitomycin C (Sigma) per milliliter with varied volumes as determined by the calculated doses of 5, 10, 15 and 20 micrograms of Mitomycin C per million cells, thus the numerical doses employed are 5-5, 5-10, 5-15 and 5-20.
Additionally, one flask which served as positive control for reversal of attenuation was treated with 150 milliliters of a solution containing 1 microgram of mitomycin C per milliliter, corresponding to numerical dose of 1-10, while one more flask treated with a solution without mitomycin C and denoted by 0-0, served as negative control for comparison.
Viable cell counts were performed on separately collected 3T3 and keratinocytes at intervals of 3 days until 9 days. For separate cell collection, the cultures were first treated with 0.02% EDTA to selectively detach 3T3 which were collected into PBS followed by collection of keratinocytes into separate vials after detaching them using 0.25% trypsin + 0.03% EDTA solution containing 0.025% glucose.
Statistics: Growth curves for various treatment groups of 3T3 and the respective keratinocytes were plotted with viable cell number on y-axis and time period on x-axis. The influence of various numerical doses on cell numbers of 3T3 and keratinocytes was statistically analyzed by Student's t-Test. A line diagram was plotted with numerical doses on x-axis and cell numbers representing 3T3 and keratinocytes on days 3, 6 and 9, respectively, on y-axis. Similarly, a scatter plot was constructed with numerical doses on x-axis and cell numbers on y-axis and separate linear trend lines representing 3T3 and keratinocytes, respectively, were plotted by calculating the least squares fit through cell count data points using regression analysis. The relationship of change in cell numbers between 3T3 and keratinocytes from all numerical doses with and without including 0-0 and 1-10 were compared by correlation coefficient.
Results and Discussion: There was significant (PK0.001) increase in 3T3 cell number in 0-0 (untreated control cells) as one would expect and those treated with 1-10 and 5-5 Mitomycin C also showed significant (P<0.01) increase in cell number compared to the 30,000 seeded cells per well, indicating that 1-10 and 5-5 were similar in exerting reversal of growth arrest. The increased cell number in the order of lower to higher cell number was 5-5 > 1-10 > 0-0 (Figure 3) which is apparently commensurate with the decrease in concentration of Mitomycin C from 5 to 0 micrograms. On the contrary, there was significant (P<.001) decrease in cell number by 5-10, 5-15 and 5-20 until day 9. This decreasing trend in cell number in the order of higher to lower cell number was 5-10 < 5-15 < 5-20 which is concomitant to the increase in numerical dose of Mitomycin C.
The attenuation reversal in 5-5 is consistent with similar observation in example-1 wherein 3T3 cells were plated alone. However, the proliferation after exposure to 5-10
micrograms Mitomycin C per milliliter in example 1 was apparent only by day 20 and hence was not observed during the 9-day period of culture when these cells were plated with keratinocytes in this example. The chances of noticing proliferating foci due to apparent attenuation failure with such a dose in co-cultures are low since longer culture times are necessary by which time most of the successfully attenuated cells are detached from the culture surface and consequently requiring replenishment with fresh stocks of attenuated 3T3. Several laboratories practice to replenish the cultures with fresh stocks after selectively detaching the leftover 3T3 cells using EDTA in order to keep the keratinocytes proliferating optimally (Daniels et al 1996). This perhaps could be the possible reason why there have been no reports on failure of growth arrest by 5 micrograms of Mitomycin C. The surviving 3T3 with recovered proliferative ability could only be detected as contaminants after about 4-6 passages of keratinocytes when they are plated without attenuated 3T3 to test for such contamination. Such recovered feeder cells were frequently observed in our laboratory along with keratinocytes which were plated alone after they were cultured for 4 passages using 3T3 feeders attenuated with a numerical dose of 5-10 Mitomycin C (Figure 4).
When such keratinocytes were serially cultured for about 6 passages using 3T3 feeders attenuated by employing 5-10 mitomycin C, rapidly growing 3T3 fibroblasts with apparent migratory patterns were observed amongst the slow growing keratinocytes that eventually became aborted (Figure 5 A and 5B). However, no such abrupt growth of 3T3 was detected in keratinocyte cultures that were serially grown for even 7 passages using 3T3 feeders attenuated with either 5-15 or 5-20 mitomycin C. The results suggest that the reverted cells arising out of the ineffectively attenuated 3T3 cells perhaps resist the mild action of EDTA which was used to selectively remove them from keratinocyte cultures, accumulate over serial passages and appear as proliferative foci after several passages. Evidently, such events were totally prevented from happening by the adoption of differential numerical dosing approach.
There was a highly significant negative correlation between cell counts of 3T3 and keratinocytes while including all numerical doses on day 3 (P<0.01), day 6 (P<0.001) and
day 9 (P<0.001). The negative correlation with 5-5, 5-10, 5-15 and 5-20 of Mitomycin C excluding 0-0 and 1-10 remained significant at P<0.01 and PO.05 for day 3 and day 6 or day 9, respectively (Figure 6) indicating that within a single concentration of 5 micrograms of Mitomycin C per milliliter, differential feeder functionality could be maneuvered through numerical dose-dependant extent of attenuation. There was overall significant negative correlation between the numbers of 3T3 and keratinocyte cells on an axis of increasing increments in all numerical doses employed over the entire period and is represented by linear trend lines with R values of 0.83 for 3T3 and 0.86 for keratinocyte cells (Figure 7).
The results prove that differential attenuation capability of any given concentration of Mitomycin C could be exerted by manipulation of doses expressed in terms of number of cells at the time of exposure in addition to its concentration expressed as weight per volume or molarity. This dose-per-cell dependant differential is not only apparent in the context of achieving irreversible growth arrest in 3T3 cells, but also in obtaining an optimal growth stimulatory influence on the target epidermal keratinocyte stem cells. The data from this experiment hint at a possibility of achieving even more optimal results in this direction by investigating a similar influence employing Mitomycin C at doses higher than 20 micrograms per million feeder cells and concentrations higher than 5 micrograms per milliliter of treating solution. Before testing this possibility on keratinocytes directly, it is essential to determine the higher concentrations to be employed on 3T3 cells.
Example 3:
Attenuation with higher numerical doses:
Experiments with only 3T3 were performed to verify the assumption that a higher than the most popularly used concentration of 5 micrograms Mitomycin C per milliliter and higher doses than 20 micrograms per million cells which was proven to be optimal in the above examples, might result in much better extent of attenuation of 3T3 cells. Hence, 3T3 cells were exposed to a 2-hour pulse of Mitomycin C at concentrations of 6, 7 and 8 micrograms per milliliter and each at doses of 10, 20 and 30 micrograms per million
cells, respectively, corresponding to numerical doses of 6-10, 6-20, 6-30, 7-10, 7-20, 7-30, 8-10, 8-20 and 8,30. The cells exposed to each numerical dose were reseeded in 24 well plates a density of 15,000 cells per square centimeter (30,000 cells per well) and viable cells were counted by Trypan blue exclusion method at the end of 3, 6 and 9 days. Untreated 3T3 cells represented as 0-0 were used as control for comparison.
Statistics: Growth curves representing viable 3T3 cell number at various numerical doses of Mitomycin C on y-axis and time periods with on x-axis were plotted. The influence of Mitomycin C on 3T3 cell number in between different time points of numerical doses was statistically analyzed by Student's t- Test.
Results and Discussion: The cell number reduction over a period of 9 days was in a numerical dose-dependant manner and the extent of reduction in the order of higher to lower cell number was 6-10 < 6-20 < 6-30 < 7-10 < 7-20 < 7-30 < 8-10 < 8-20 < 8-30 (Figure 8). The differential action of various numerical doses falling under 6 micrograms was not apparent at 3 days post-plating time point as revealed by a more or less uniform loss of cells. Although, the differential action was significantly appreciable by 6 days but it was particularly abolished in between 6-20 and 6-30 by 9 days suggesting the need to test higher workable doses than 30 micrograms per million cells within 6 microgram concentration. Further, the number of 3T3 cells exposed to any of the numerical doses within 8 micrograms was about 50 percent or less than the seeded cell number as early as 3 days in culture, and the numerical doses within 7 micrograms brought a similar influence in 6 days. It is noteworthy that only about 20 percent or less of seeded cells remained live with 7 and 8 micrograms by the end of 9 days. Additionally, it was found that exposure to 8 micrograms resulted in 7% loss of 3T3 cells by the end of 2-hours pulsed exposure with the remaining cells becoming rounded and attaching only thereafter indicating acute toxicity. Such a quick loss of growth-arrested feeder cells is highly disadvantageous to the growth of keratinocyte stem cells requiring re-plating of keratinocyte-cultures with fresh stocks of feeders. Hence, employment of higher Mitomycin C concentrations than 7 micrograms will not be advantageous for optimal keratinocyte growth. Therefore, it is essential to evaluate the growth potential of
keratinocyte stem cells using 3T3 cells attenuated with concentrations of 5 and 6 micrograms Mitomycin C at doses of 20, 40, 60 micrograms per million cells employing higher seeding density of 3T3 feeders to ensure presence of sufficient number of feeders for persistent target cell stimulation. Higher doses than 60 micrograms per million cells are not practically convenient because of the limitation of culture flasks to take the requisite larger volumes of medium with the lower Mitomycin C concentration of 5 micrograms per milliliter.
Example 4:
Optimal growth stimulation of keratinocyte stem cells:
In order to estimate the ability of differentially growth arrested 3T3 cells by numerical doses of 5-20, 5-40, 5-60, 6-20, 6-40, and 6-60 in supporting the growth of target stem cells, experiments were conducted by seeding into 24 well plates the primary human epidermal keratinocytes (Genlantis, USA www.genlantis.com) and differentially growth arrested 3T3 cells at densities of 5000 (10,000 per well) and 25000 cells per square centimeter (50,000 per well), respectively. Additionally, untreated 3T3 cells as denoted by 0-0 and those treated with a numerical dose of 7-20 were included as negative and positive controls, respectively, for comparison. The cells were grown in KCM with a change of medium every alternate day. The attenuation protocol of 3T3 was as per example 1. Viable cell counts were performed as per example 2 on separately collected 3T3 and keratinocytes at intervals of 3 days until 9 days. For separate cell collection, the cultures were first treated with 0.02 percent EDTA to selectively detach 3T3 which were collected into PBS followed by collection of keratinocytes into separate vials after detaching them using 0.25 percent trypsin + 0.03 percent EDTA solution containing 0.025 percent glucose. Additionally, the keratinocyte cell cultures were serially sub-cultured 7 times employing each numerical dosing approach to look for any contamination by the reverted 3T3 cells.
Statistics: Growth curves for various treatment groups of 3T3 and the respective keratinocytes were plotted with viable cell number on y-axis and time period on x-axis. The influence of various numerical doses on cell numbers of 3T3 and keratinocytes was
statistically analyzed by Student's t-Test. A line diagram was plotted with numerical doses on x-axis and cell numbers representing 3T3 and keratinocytes for days 3, 6 and 9, respectively, on y-axis. Similarly, a scatter plot was constructed with numerical doses on x-axis and cell numbers on y-axis and separate linear trend lines representing 3T3 and keratinocytes, respectively, were plotted by calculating the least squares fit through cell count data points using regression analysis. The changes in cell number between 3T3 and keratinocytes from all numerical doses with and without including 0-0 (control) were compared by correlation coefficient. The influence of Mitomycin C on 3T3 cell number in between different time points, concentrations and doses was statistically analyzed by Student's t-Test.
Results and Discussion: There was a highly significant negative correlation (P<0.001) between the cell numbers of 3T3 and keratinocytes while including all cell count-data for Days 3, 6, 9 or 12 (Figure 10) indicating that the superior growth supporting activity of 3T3 feeders could be achievable with concomitant sequential increase in the extent of attenuation accomplished through differential numerical dosing. However, the negative correlation was insignificant when calculated independently for days 3, 9 or 12, while it was significant (P<0.05) for day 6 indicating that optimal growth of keratinocyte cells could be achieved at around 6 days of culture under the present culture conditions. Correspondingly, the polynomial trend line exhibited a peaking tendency for keratinocyte cell number at 9 days time point of co-culture with feeders and thereafter exhibited a downward slope until 121 day, by which time 3T3 cells from all the differentially attenuated stocks were almost reduced to bare minimum. Therefore, it may be inferred that in case the keratinocyte stem cells are to be cultured using these feeders for more than 6 days, replenishing the cultures with fresh attenuated feeders becomes necessary. The calculated R2 values for 3T3 and keratinocyte stem cells were 0.99 and 0.75, respectively after excluding the negative control (0-0) values.
The smaller isolated curves drawn to represent the progress of cell number at each time point (Figure 10) showed a characteristic peak of growth for keratinocyte cells at 5-60. Subsequently, the keratinocyte cell number significantly (P<0.05) sloped down from 6-20
and beyond at 3 days time point. However, the cell number at 6-40 on days 6 and 9 showed significant (P<0.02) recovery to almost as much as it was at 5-60. Further, there was more than one and half times higher yield of keratinocyte cells with 5-60 attenuated feeders than with 5-20 after a 6-days period of co-culture. These findings in our experimental set-up demonstrate that employing a concentration of 6 micrograms Mitomycin C per milliliter is not of any additional advantage and optimal feeder functionality in the present clone of 3T3 was achieved by a concentration of 5 micrograms Mitomycin C per milliliter itself, but at a dose of 60 micrograms Mitomycin C per million cells. Additionally, after 6 days in culture the keratinocytes in several colonies appeared squeezed possibly because of obstruction imposed by the broader and firmly attached 3T3 cells attenuated by employing 5-20 Mitomycin C (Figure 9A), which is in contrast to the loosely anchored and degenerating 3T3 cells attenuated by 5-60 Mitomycin C (Figure 9B) that would perhaps promptly leave the culture surface allowing uninhibited spread, proliferation and migration of keratinocytes. Higher than 60 micrograms Mitomycin C per million cells may not be practicable to work out because of large volume requirements, which is not convenient with the currently available culture flasks. The keratinocyte cell cultures serially sub-cultured 7 times employing each numerical dosing approach revealed no proliferation foci in any of the numerical doses employed in this experiment further proving the usefulness of the differential attenuation protocol.
The deciding factors for volume fixation are the exposure cell number, concentration and dose and the exposure cell number in all flasks to be treated as a single batch should be invariably the same for consistency of results. As per our experience an exposure cell number of 200,000 cells per square centimeter could be obtained reproducibly with a small deviation of less than 2 percent within a single batch of feeder cells generated on any given occasion. This consistency is necessary since the numerical dose fixation before exposure to Mitomycin C is based on cell counts performed on parallel flasks and is primarily dependant on exposure cell number.
Experiment 5:
Test for irreversibility:
The 3T3 cells differentially attenuated with numerical doses of 5-20, 5-40, 5-60, 6-20, 6-40, 6-60 and 7-20 Mitomycin C as described in example 4 along with those attenuated by the numerical dose of 5-10 Mitomycin C were plated in 75 square centimeter flask each (Nunc), at a seeding density of 35,000 cells per square centimeter in KCM. Similarly, each of the differentially attenuated 3T3 cells was seeded into 24 well plates in triplicate wells at a density of 25,000 cells per square centimeter. The cultures were maintained at standard incubating conditions with regular medium changes and three independent observers assessed the weekly regression in cell density by assigning grades between five and zero, representing highest density as on day-one and no attached cells on plastic surface, respectively. The cultures were maintained until all the cells degenerated so as to verify the irreversibility of attenuation.
Results and Discussion: The cultures maintained in 24-well plates exhibited signs of deterioration within the first week of seeding and the degree of more extensive degeneration to less was in order of 7-20>6-60>6-40>6-20>560>5-40>5-20, while in 5-10 the disintegrated cells were rare by this time but appeared to increase thereafter and distinctively apparent only after 2 weeks (Figure 11 A). Eventually, 4 to 10 foci of proliferation activity per well were noticeable during 3rd week and some of them remained stable, while the remaining continued to grow until confluence in about 3-6 weeks time. On the contrary, no intact cells were noticeable after 2 weeks period in 7-20, 6-60 and 6-40 followed by 6-20, 5-60 and 5-40 until 3 weeks time and all cells disappeared in 5-20 only after 4th week.
The attenuated 3T3 cell cultures maintained in flasks exhibited first signs of deterioration in the same decreasing order as in well plates. However, the rate of disintegration of 3T3 cells in 75 cm flasks was evidently slower than in wells, which is commensurate with higher seeding density of 35,000 cells per cm2 (Figure 1 1B).
There were no noticeable intact cells after 3 weeks of incubation of 7-20, 6-60 and 6-40 Mitomycin C treated cells followed by 6-20, 5-60, 5-40 and 5-20 at the end of 4 weeks. The difference in the extent of disintegration between arithmetically maneuvered doses of 20 and 60 micrograms Mitomycin C per million cells within the concentration of 5 micrograms Mitomycin C per milliliter is well appreciable (Figure 12 A and B). Conversely, the reversal of 3T3 cells that were growth arrested with 5-10 Mitomycin C was similarly detected in culture flask as in well plates, but a very higher incidence of proliferative foci of morphologically distinct clones was observed (Figure 13) and reached confluence in about 8-12 weeks.

We claim:
1. A culture system for the growth of stem cells comprising,
a. inactivating the feeder cells effectively with Mitomycin C; and
b. providing a substrate in the form of such feeder cells;
c. achieving effective feeder cell growth arrest in terms of permanent loss of
growth in them while maximally stimulating epidermal keratinocyte stem
cell growth;
characterized in that the concentration of mitomycin C is titrated with respect to the number of cells.
2. A culture system as claimed in claim 1, wherein the stem cells are epidermal keratinocyte stem cells and the feeder cells are any feeder cell.
3. A culture system as claimed in claim 1, the feeder cells are murine embryonic dermal fibroblast 3T3 cells
4. A culture system as claimed in claim 1, wherein, the concentration of Mitomycin C solution is per million feeder cells.
5. A culture system as claimed in claim 1, wherein the feeder cells are exposed to Mitomycin C in a pulsed manner.
6. A culture system as claimed in claim 3, wherein the exposure to Mitomycin C is in one to three hour pulse, preferentially a two hour pulse.
7. A culture system as claimed in claim 1, wherein the concentration of mitomycin C is in the range of 5 to 8 micrograms per milliliter and the dose is in the range of 5 to 60 micrograms per million cells.

8. A culture system as claimed in claim 1, wherein the concentration of mitomycin C
is 5 micrograms per milliliter and the dose is 60 micrograms per million cells.
9. A culture system as claimed in claim 1, wherein the concentration of mitomycin
C is 5 micrograms per milliliter and the doses are 5 and 10 micrograms per
million cells, which produces reversible growth arrest.
10. A culture system as claimed in claim 1, wherein the feeder cells are murine
embryonic dermal fibroblast 3T3 cells.
11. A culture system for maintaining the growth of epidermal keratinocyte stem cells
substantially as herein described with reference to forgoing examples and
drawings.