An Efficient Method for Rapid Establishment of Fibroblast-Like Cells from Horse Subcutaneous Adipose Tissue

Research Article
An Efficient Method for Rapid Establishment of Fibroblast-Like Cells from Horse Subcutaneous Adipose Tissue

Corresponding author: Dr. M.Quamrul Islam, CellAmp Laboratories,Fanjunkaregatan 86,S-582 16 Linköping, Sweden, Tel : +46-0739196546; E-mail: quis@cellamplabs.com

Abstract

Adipose-tissue derived stromal cells (ADSCs) are easily obtainable multipotent mesenchymal stem cells capable of differentiation into various cell lineages making it an attractive cell source for regenerative medicine. ADSC is generally isolated from subcutaneous adipose tissue by enzymatic digestion of biopsies followed by removal of undigested tissues and seeding of the dissociated cells onto plastic. This is an inefficient procedure where only a tiny fraction of cells are established in culture and rest of the cells are lost. Development of new methods would be desirable to recover most cells of the originally collected biopsies. This would reduce the time between biopsy collection and transplantation by producing large quantities of cells within a short time span. One of the benefits of this method would be to provide autologous cells for quick replacement therapies. Present report describes a new cell culture method which allows establishment of almost all cells of adipose tissue biopsies on plastics and the adherent cells show fibroblast-like morphology with rapid self-renewal capacity. ADSCs of subcutaneous fat tissue of horse can be isolated efficiently by mincing the excised biopsy with a surgical scissor and culturing of fragmented tissue in RPMI 1640 medium containing 15% ordinary fetal bovine serum for 2-3 weeks without change of medium. The conditioned media of highly proliferating ADSCs not only maintain self-renewal but also improve proliferation of other cells indicating that proliferating ADSCs secrete growth promoting factors in the culture media. The new method has direct application both in basic research and in regenerative medicine. The new method is named as “conditioned media-mediated Cell Amplification, (cmCellAmp)”.

Keywords: Adipose-Tissue Derived Stromal Cell; Adult Stem Cell; Embryonic Stem Cell; Autologous Transplantation; Immortality; Conditioned Media; Fibroblast; Cell Multiplication; Method.

Introduction

MStem cells are defined as cells with extended self-renewal capacity and ability to differentiate into cells of multiple lineages under appropriate signals [1-3]. There are two major types of stem cells: embryonic and somatic. The embryonic stem cells are derived from the inner cell mass of pre-implantation blastocyst. The ES cells have extensive self-renewal capacity and almost all types of body cells can be produced by ES cells making them ideal for cell replacement therapies [4]. However, their human applications raise both ethical and biological concerns including the problems of immune rejection and tumor development. On the other hand, somatic stem cells exist in almost all organs of both new born and adults making them suitable cell source for autologous transplantation in human cell-based therapies [5-7]. It has been proposed that the in vivo existence of somatic stem cells is rare [8]. Therefore, it is time consuming to generate large numbers of somatic stem cells for clinical applications from a very few originally isolated cells. In addition, self-renewal capacity of somatic stem cells found to be diminished during in vitro cultivation because of repeated divisions [9, 10]. Despite limited self-renewal and restricted differentiation, somatic stem cells hold great promise for treating human degenerative diseases because of the prospect of deriving patient- specific somatic stem cells for the replacement of damaged tissue by autologous cell transplantation [11].

Somatic stem cells can be obtained from diverse tissues including bone marrow, adipose tissue, skeletal muscle, umbilical cord blood, peripheral blood, skin tissue, and trabecular bone [12, 13]. These plastic-adherent fibroblast-like cells are called as mesenchymal stem cells [14, 15] and they show similar phenotypic features regardless of their tissue origins [16- 19]. In addition, MSCs can produce cells of both mesenchymal and non-mesenchymal tissues upon induced differentiation in vitro and spontaneous differentiation in vivo. Considering easy accessibility and abundance, the adipose tissue is becoming an attractive source of MSC [20-23]. Also, collection of a subcutaneous adipose tissue biopsy is less painful than a bone marrow aspiration [24-26]. Isolation of MSCs from adipose stromal tissue is commonly done by enzymatic digestion of biopsies followed by removal of undigested tissue fragments and finally seeding of the dissociated single cells onto plastic to allow the proliferation of viable cells [27-29]. During this procedure most of the cells are lost and less than 1% of the original cells actually represent any MSC culture [24-30]. Therefore, development  of new methods would be highly desirable in order to recover most cells of the originally collected biopsies. This would reduce the time between collection of biopsy and transplantation by producing large numbers of cells within a short time span [23, 31]. One of the benefits of this method would be to provide autologous cells for quick replacement therapies. Present communication describes a new cell culture method which allows establishment of almost all cells of adipose tissue biopsies and the established plastic-adherent cells show fibroblast- like morphology with rapid self-renewal capacity.

Materials and Methods

Isolation of subcutaneous adipose stromal cells

Adipose tissue stromal cells (ADSCs) were isolated from subcutaneous fat of horse. Briefly, fat skin biopsies were taken from the lower side of tail base (kindly provided by Elisabet Roman Granath, ATG Hästklinik, Mantorp, Sweden). Hair-free stromal tissue along with skin were immediately placed into a 50 ml Falcon tube containing 5 ml serum-free RPMI 1640 medium (Invitrogen) supplemented with 2% penicillin-streptomycin. The tubes were kept at room temperature in the horse clinic for an hour and they were transported to the tissue culture laboratory within the next hour by a car. After discarding media, a tissue sample was removed from the tube into a 100 mm Falcon tissue culture dish, after careful removal of black skin, small pieces were made by sterile surgical scissors. A small piece of blood-free stromal tissue of approximately 0.5 cm2 size was transferred to a new 100 mm Falcon dish with one drop of RPMI medium and finely minced into small pieces. The whole content of the chopped tissue was mixed several times with 6 ml RPMI medium supplemented with 15% FBS and 1% penicillin-streptomycin (complete RPMI) by a pipette and transferred into a 75 cm2 Falcon cell culture flask. Any remaining cell fragment in the Petri dish was transferred to the same flask by mixing with additional quantity of media to make a final volume of 16 ml. The culture flask was then incubated at 37oC with 5% CO2 and 80% relative humidity. The incubated flasks were examined daily with an inverted microscope to see cell attachment and recording the morphology of cells. No media change was made until the attached cells became confluent.

Cell passaging

Before passaging cells, the entire 16 ml conditioned media (CM) of a confluent culture flask was collected in a tube along with unattached cells and tissue fragments. The attached cells on plastic surface were then washed with 3 ml trypisin (1X PBS-trypsin, PAA) by incubating at 37oC for 2-3 minutes. After discarding the washing trypsin, 1 ml trypsin-EDTA (GibcoTMInvitrogen Corporation, Paisley, Scotland, UK) solution was added to the flask and incubated again at 37oC for additional 2-3 minutes to detach cells from culture flask. The detached cells were then suspended in previously collected 16 ml CM and divided into four equal parts to plate into four Falcon 75 cm2 flasks, 4 ml each. A total volume of 16 ml medium was made in each flask by addition of 12 ml complete RPMI. The medium was not changed until cells reached confluency. Confluent cells were passaged into new flasks with a split ratio of 1:4 using same procedure as mentioned above. Excess cells were cryopreserved.

Collection of conditioned media and storage

The CM of ADSCs were collected from highly proliferative cells at high density, usually 4-5 days after passaging cells into new flasks when the CM became thick and slightly yellow. In some cases, fresh medium was conditioned overnight by proliferating cells of near confluent cultures before colleting slightly yellow CM. It should be emphasized that from a high density culture of 75 cm2 flask approximately 16 ml CM could collected each time. Freshly collected CM was used directly after passing through membrane filters (0.2 mm pore size). For long-term use, the CM was frozen at -70oC immediately after collection without filtering. The frozen CM was thawed in a 37oC water bath and centrifuged to remove cellular debris prior to use. For the recovery of cells from liquid nitrogen and passaging of cultured cells, diluted CM was used by mixing 1 part CM with 2 parts fresh complete RPMI. To transform cells only freshly collected, filter-sterile undiluted CM was used.

Shifting of Cells in DMEM to Induce Aging

Parallel cultures of ADSCs were established by shifting cells of passage 3 from RPMI to Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose) supplemented with 15% Fetal Bovine Serum (FBS), 1% MEM nonessential amino acids, 1% penicillin- streptomycin (hereafter complete DMEM). Some ADSC cultures were maintained under infrequent media changes conditions in order to induce in vitro cellular aging [32]. Tissue culture reagents were obtained from PAA laboratories GmbH (Pasching, Austria), if not stated otherwise.

Photomicrograph and image recording

Cell morphology was recorded by using an inverted microscope with 10X objective (AXIOVERT, Zeiss) equipped with a Canon digital camera (Power Shot A95) setting the zoom at 4.9X and all images composed in Adobe Illustrator 10.

Results

Poor self-renewal of ADSCs grown by regular changes of media

In the present study, ADSCs were isolated from horse subcutaneous stromal tissue without using any enzyme treatment. Fatfree stromal tissue of an approximate size of 0.5 cm2 was finely minced with surgical scissors and the entire mass of minced
tissue was plated into a culture 75 cm2 flask. Microscopic examination of freshly plated stromal tissues showed fibrous structures with no visible sign of living cells (Figure 1A). The culture flasks were examined under phase contrast microscope on daily basis to find adherent cells on plastic but no cell was adhered until day 4. The first medium change was made at day 6 from the initiation of the culture and thereafter medium was renewed every 3-4 days. During first medium change, most of the suspended cells and tissue fragments were lost along with the original medium. Gradually, adherent cells formed colonies with a brown body at the centre of each colony and spindle shaped cells could be found in the periphery of the colonies (Figure 1B). Subsequently, the brown body in the centre of the colony disappeared by spreading cells on plastic (Figure 1C). Although sporadic rounded mitotic cells could be observed in the culture flasks, in general multiplication of the ADSC cells was not significantly improved even after regular changes of media (Figure 1D). This result indicates that the addition of fresh serum growth factors through medium change has limited effect on the self-renewal of ADSC isolated by no enzyme treatment.

Figure1. Isolation of adipose tissue stromal cells (ADSCs) by mincing horse skin: Culture flasks of ADSCs (A) unattached tissue fragments at day 5, (B) a small group of adherent cells after the removal of medium at day 7, (C) the attached cells at day 9 and (D) small colony of cells at day12. Medium changes were made every third day. Bars: 100 mm.

Persistent self-renewal of ADSCs in the absence of medium change

To determine whether the retention of original media in the same culture flask for prolonged period improve self-renewal, parallel cultures of ADSCs were established where no medium change was made after the initiation of culture. Microscopic examinations of these cultures at day 8 showed large numbers of attached cells, in contrast to the culture flasks where medium was changed at day 6. Regular monitoring of the culture flasks, where no media changes were made, showed large number of attached cells with the increase of culture period, a partial view of a culture flask of ADSC, where a large fragment of partially attached stromal tissue with many spindle shaped cells could be seen beneath the brown body (Figure 2A). Interestingly, attached cells in the beginning showed irregular outlines covering large areas (Figure 2B). Gradually, these cells shrunk and converted into spindle shaped cells through morphological changes. The spindle shaped cells frequently found to be undergoing mitotic division (Figure 2C). The numbers of mitotic cells steadily increased until the culture became confluent. It should be noted here that larger tissue fragments could be observed as completely suspended objects or as partly adherent object on plastic surface even in the confluent cultures (not shown). With the increase of incubation period, exceeding 10 days, the color of the conditioned media started to change and turning into yellower. During this phase of culture, many partially attached large tissue fragments adhered completely (Figure 2 D), even though many non-adhered tissue fragments still be present in the culture. The non-adherent tissue fragments could be transferred along with the old medium into a new culture flask and the previously unattached stromal tissue subsequently found to be attached on plastic. It should be noted here that no cell death was observed at any stage of this procedure and ultimately most of the cells of the originally dissected tissue could successfully be attached by carrying floated cells into new culture flasks. However, adequate numbers of ADSCs could be obtained from the first culture flask without the need of making secondary or tertiary culture flasks. Together, these results indicate that the isolation of ADSCs without using enzyme treatment is an efficient method to establish them in culture without any cell loss. Moreover, culture of the ADSCs in the absence of medium change is a valuable method for their multiplication through the use of CM.

Figure 2. Establishment of adipose tissue stromal cells (ADSCs) from horse skin with no enzymatic treatment and no medium change (A) a partial view of a culture flask at day 8, (B) at day 10 (arrows indicate flat cells), (C) high mitotic activities at day 12 (arrows indicate rare flat cells), and (D) at day 15 showing numerous mitotic cells. Note the fibroblastic morphology of ADSCs.. Bars 100: mm.

The above experiment was repeated three times with adipose stromal tissues derived from three independent animals and the results were similar. It should be noted that the excised tissues were preserved at room temperature for not longer than 8 hours before culturing was done. In the modified experiments, where the excised tissues were preserved in refrigerators, the number of attached cells found to be reduced. Also, longer preservation (more than 24 hours) even in room temperature severely changed the capacity of cells to attach on plastic (Table 1). These results indicate that the excised adipose tissues must be preserved in room temperature and the culture of these tissues must be initiated as soon as possible to get the majority attached cells in shortest possible time.

Table 1. Effect of storage conditions of adipose tissue stromal cell biopsies on subsequent cell attachment.

Selective cell death of ADSC culture with no medium change for prolonged period

Maintenance of confluent culture of ADSCs exceeding 21 days showed localized cell death, although some cells found to be dividing under the same condition (Figure 3A). With the increase of culture period the conditioned media of these cultures display yellowish colours and the dead cells were aggregated in many places (Figure 3B). Later, the aggregates of dead cells detached from the surface and empty spaces were created in the culture flasks (Figure 3 C, D). Eventually, the empty spaces were filled by increasing the number of surviving cells. These results may indicate that apoptosis was not due to general nutritional deficiency of culture medium rather only the sensitive cells might have died due to presence of certain cell products in the culture media or pH changes of culture flask.

Interestingly, when the old growth media of the partially dead cultures was replaced completely with fresh media then morphology of attached cells was drastically changed showing a pattern of allied cells similar to myogenic-like differentiated cells in culture (Figure 3E, F). The spontaneously differentiated- like associated cells showed homogeneous morphology through out the culture flask. There was no visible mitotic activity anywhere in the culture flask and this growth arrested state of cells could be reversed and proliferative cultures could be established by passaging them into new culture flasks with growth media containing 25% CM of proliferating ADSCs and 75% fresh media. It should be emphasized that replacement of old media with new one did not improve the proliferation of  partially dead cultures without passaging them in new culture flask. This is true even in the case of highly proliferative ADSCs where renewal of medium was found to be not beneficial for cell proliferation. Therefore, whenever medium changes were required cells were passaged into new culture flasks with 25% CM and 75% fresh medium and no medium change was made until the culture was nearly confluent.


Figure 3. ADSCs grown long times with no change of media showing cell death as well as mitotic cells: (A-D) culture flask at day 22 showing elongated healthy cells and localized cell deaths (brown patches) and mitotic cells (arrows), (C, D) continued proliferation of cells at 22 days (arrows indicate mitotic cells), (E) a strip of dead cells in the background of healthy cells, and (F) change of old medium with fresh medium resulted myogenic-like differentiated cells. Bars: 100 mm.

Culture of ADSCs in DMEM without conditioned media showed senescent-like cells

Passaging of near confluent cultures of highly proliferating ADSCs with the split ratios of 1:4 in the presence of CM maintained high replication rates at least up to 36 passages (Figures 4 A, C, E). Since the conventional culture of ADSCs is done with DMEM and various concentrations of fetal bovine serum, ADSCs of present study at passage 3 were shifted to fresh DMEM without addition of CM. Although shifting of ADSCs in DMEM initially maintained mitotic activity but gradually shifted cells became flat (Figure 4D). Continuous culture of the ADSCs in DMEM without addition of CM decreased mitotic cells and after 7 to 10 passages in DMEM all mitotic activities were stopped showing morphological signs of cellular senescence (Figure 4 F). This result indicates that the morphological cellular senescence- like phenotype of ADSCs may be caused by continuous absence of certain factors in the culture media and these missing factors can be supplied by proliferating ADSCs and that the CM of these cells can prevent induction of morphological cellular senescence-like phenotype.


Figure 4. ADSCs grown in DMEM with no addition of CM resulted cellular aging: (A) highly replicating ADSCs at passage 3 grown with CM, (C) at passage 12, (E) at passage 17, (B) ADSCs at passage 4 grown for 24 hours in complete DMEM but without CM, showing more flattened cells (D) at passage 12 grown with no CM showing cellular aging, and (F) at passage 13 grown with no CM shows no mitotic activity. Note the high mitotic activity of ADSCs grown in the continuous presence of CM, irrespective of their passage numbers (A, C and E). Bars: 100 mm.

Morphology of epidermal cells can be changed into fibroblast- like cells by treatment of conditioned media derived from ADSCs

Cells of the epidermal layer of horse black skin were isolated by careful removal of stromal layer. Culture of partially separated black skin tissue generated mixed culture of epidermal cells and fibroblastic cells. The mixed culture had multilayered fibroblast-like cells with many dividing cells and a single layered epidermal cells with non-dividing cells (Figure 5A). Cells of mixed cultures were detached by repeated and prolonged treatment of trypsin. The detached cells were then carefully mixed and plated them into several culture flasks with growth medium containing CM derived from highly proliferating ADSCs.  The plated cells showed mostly fibroblast-like cells in the background of some patches of non-fibroblastic cubical cells (Figure 5 B-E). Subsequent passaging of these cells in new culture flasks with continuous treatment of CM produced morphologically transformed uniform culture of fibroblastic cells, similar to ADSC-like morphology (Figure 5 F).

Figure 5. Culture of black skin tissue of horse generates mixed culture of epidermal cells and fibroblastic cells and the morphology of epidermal cells can be altered into fibroblast-like cells by treatment of fresh CM: (A) mixed culture of fibroblast-like cells and epidermal cells (arrows indicate the border between two cell types) in growth medium containing fresh CM. (B-E) mixed cells display patches of cubical cells (indicated by arrows) in the background of fibroblastic cells, (F) subsequent passages of these cells produced solely of fibroblastic cells. Bar: 100 mm.

To exclude the possibility that the mixed culture had only selectively multiplied fibroblast-like cells, pure epidermal cells were isolated from monolayer of Petri dishes using cloning rings. Culture of these cells for 2-3 days in regular medium showed exclusively a single layer of flat epidermal cells of large sizes with no mitotic activity (Figure 6 A, B). The isolated epidermal cells were then treated with freshly collected, filter- sterile, undiluted CM derived from highly proliferating ADSCs. Within 24 hours of addition of CM, non-dividing epidermal cells were transformed into fibroblast-like cells and eventually they produced a homogeneous culture of highly proliferating cells and these cells maintained fibroblast morphology when maintained in culture with no medium change (Figure 6 C, D). These results indicate that fibroblast-like ADSCs produce celltype specific factors in the conditioned media which have the ability to transform epidermal cells into fibroblast-like cells.

Figure 6. Isolation of pure population of epidermal cells by ring cloning and subsequent treatment of these cells with fresh CM transformed them into fibroblastic cells: (A, B) flat epidermal cells, (C, D) medium containing fresh CM converted them into fibroblastic cells in four days, retention of the CM in the same flask eventually transformed them into pure ADSC-like cells.Bar: 100 mm.


Conventional procedure for the isolation of adipose stromal cells (ADSCs) is by digesting adipose tissue with collagenase and plating of enzyme dissociated cells in appropriate culture medium after removal of undigested tissue fragments. This procedure permits only a tiny fraction of original cells to be established in culture. The poor yield of this procedure is probably caused by physical damage of cells due to enzyme treatment resulting poor viability of proliferation competent cells. Alternatively, enzyme dissociated cells may not grow in isolation as single cells. Attempts have been made to isolate adipose stromal cells by mincing tissues without enzyme treatment and plating of dissociated cells on plastic with regular changes of medium from culture flasks. However, yield of attached cells is not substantially increased may be because of loss of non-adherent cells and tissue fragments during medium changes.

The present study demonstrates that the adipose stromal cells of subcutaneous fat tissue can be isolated efficiently by mincing excised tissue with a surgical scissor and culturing of fragmented tissue in RPMI 1640 medium containing 15% ordinary fetal bovine serum for 2-3 weeks without change of medium. It appears that the exclusion of enzyme treatment allows survival of most cells of the tissue and non renewal of media from culture flask prevents the loss of non-adherent cells and tissue fragments. The combined effects of these two modifications from the conventional technique dramatically increase the yield of attached cells with remarkable improvement of their proliferation. The present study also shows that there is a positive correlation between incubation period of ADSC culture and prevalence of mitotic cells. It is possible that in the absence of media change, diffusible factors produced by ADSCs, gradually accumulate in the media and the concentrations of these factors continue to rise with time. These secreted factors may act in a dose dependent manner to induce proliferation of cultured cells and that is probably why the older cultures show more mitotic cells than the newer cultures. Alternatively, it is possible that in the older culture contain more cells and they produce more quantity of diffusible factors per unit time and that the increased cell density results increased production of growth promoting factors which may induce self-renewal in a concentration dependent manner.

Interestingly, freshly cultured minced adipose stromal tissues under phase-contrast microscopic exhibit thread like structure with no indication of living cell. The submerged adipose stromal tissue remain completely unattached in the culture media at least for the next 3-4 days indicating that these are not ordinary fat cells which normally float on the surface of culture medium. Afterwards, a few cells of suspended tissue begin to anchor on plastic with major portion of the tissue still remain unattached. Cells spread on large surface area in the beginning of attachment and gradually they contract producing spindle shaped cells. The morphologically altered spindle shaped cells frequently undergo mitotic division. The prevalence of attached cells increases with the prolongation of incubation period of the culture flask and ultimately all cells of suspended tissue adhere on plastics during serial culture. It should be noted that long-time preservation of adipose tissue in the refrigerator prior to initiation of culture has adverse effect on subsequent adherence of cells on plastic. Storage time exceeding 24 hours even in room temperature can change cells permanently resulting poor or no cell attachment (Table 1). These observations indicate that the survival of adipose cells prior to culture, measured by their subsequent attachment on plastics, is largely dependent on storage temperature and duration of storage. It is likely that ADSCs may not have proliferative capacity in vivo under normal conditions. They may gain proliferative capacity in vitro culture by serum growth factor stimulation and subsequently proliferative cells release growth promoting factors in the culture media. The combined effects of serum growth factor stimulation and secretion of own growth factors ultimately drive the cultured ADSCs to proliferate. It is seems that the secreted growth factors initiate self-renewal only when the concentrations of the diffusible factors reach to a critical level and that is why at least 3-4 days are required to start attachment of cells and their subsequent proliferation. However, if the biopsy is stored for longer time, exceeding 72 hours, even in room temperature cells do not respond to serum growth factor stimulation, as a result they fail to attach (Table 1).

It is known that cultured cells derived from adipose tissue produce various types of cytokines, growth factors and survival factors [26]. It has been suggested that the adipose tissue in vivo functions as endocrine organ producing various types of hormones and cytokines [32-34]. It should be noted here that the adipose stromal cells of present study were established in culture by mechanical mincing of tissues without collagenase treatment. It is likely that the exclusion of enzyme treatment preserves the native structure of adipose tissue fairly intact enabling them to produce various types of soluble factors in response to serum growth factor stimulation. Establishment of limited number of ADSCs by standard procedure of ADSC isolation by enzymatic treatment may be because of breakdown of tissue architecture as a result they fail to secrete any growth factor. In addition, frequent changes of media from the culture flasks remove even the residual tissue fragments.

It is widely believed that cultured cells require regular changes of media in order to remove toxic products of the cells and supply essential new nutrients to the cultured cells through fresh media. In this respect, accelerated proliferation of ADSCs of present study in the absence of medium change does not support this notion. Interestingly, CM of highly proliferating ADSCs not only sustain self-renewal but also improve proliferation of other cells indicating that proliferating ADSCs secrete growth promoting factors in the culture media. More spectacular finding of the present study is the ability of ADSC-derived CM to morphologically transform skin epidermal cells into fibroblast- like cells. This result indicates that the CM of ADSC contain cell type specific factor(s) which act dominantly on treated cells to convert their morphology into fibroblast-like cells, regardless the morphology of pre-treated cells.

Commonly, cultured cells are passaged regularly into new flasks when their density becomes high to maintain continuous proliferation. In contrast, the present study shows that ADSCs grow better at higher densities. Relentless mitotic activities of these cells can be maintained by passaging of confluent culture with a split ratio of 1:4 in the presence of 25% own CM and 75% fresh media. This result suggests that the self-renewal of ADSCs is influenced by direct interaction between adjoining cells through an autocrine-like signaling. Interestingly, prolonged culture of over-confluent ADSCs without medium change never results systematic cell death. Instead, sporadic cell death occurs in localized areas producing brown debris with simultaneous presence of mitotic cells in the same culture. This may indicate that cell death may not be the result of nutritional deficiency in general but probably due to over-production of certain factor(s) in the media. Alternatively, pH change may have adverse affect on the survival of some cells, as older cultures show yellowish media colour. Surviving cells of these nutritionally deficient cultures can generate highly proliferative cultures when transferred into new culture flasks with 1:4 split ratio in 75% fresh medium supplemented with 25% CM derived from highly proliferating ADSCs. However, sudden replacement of entire media of an over-confluent culture, maintained for prolonged period, with fresh media occasionally make morphological changes in the cells mimicking myogenic-like differentiation, instead of improving proliferation (Figure 3 E, F). Although no functional analysis of proliferative ADSCs of present study have not yet been made, uniform fibroblast-like morphology of proliferating cells and the homogeneous characteristic of spontaneously differentiated myogenic-like cells indicate that most of the cells have uniform capacity for both proliferation and differentiation.

We have developed a two-step cell culture protocol to improve the proliferation of normal somatic cells derived from diverse mammalian species [35, 36]. In the first step of this method, normal somatic cells are fused with the immortal mouse cell line GM05267 and immortal hybrid cells are generated containing polyploid somatic cell chromosomes. In the second step, CM are collected from the highly proliferating hybrid cells and subsequent cultivation of normal somatic cells in the presence of CM derived from hybrids allows generation of long-term growing somatic cell lines containing diploid chromosomes. Recently, we have reported reprogram of non-replicating somatic cells for long-term proliferation by temporary cell-cell contact without the transfer of any genetic or cytoplasmic factor [37]. By short time co-culture of the immortal mouse cell line GMO5267-F7 with non-replicative human mesenchymal stem cells induce proliferation of non-replicative human cells and produce highly proliferating clonal cell lines. The CM derived from GMO5267-F7 cells supports the proliferation of epigenetically reprogrammed human cells indicating the cross-species activities of CM of mouse cells. We have also demonstrated that fusion of mouse GMO5267-F7 cell line with non-replicative normal cells of various species generate immortal hybrid cells [37]. These hybrid cells can provide large quantity of conditioned media to support the proliferation of normal cells of various species [35, 36]. In this respect, CM of ADSCs will be a new tool for large scale multiplication of adult stem cells.

The CM of immortal embryonic carcinoma cells found to support the establishment of embryonic stem cells in vitro. Interestingly, trypsin treated embryonic stem cells grown in standard ES cell media do not replicate in solitary conditions. However, if these cells are grown in presence of feeder layer of early passage embryonic fibroblast they can produce colonies originating from single cells [38,39]. Clonal cell lines can also be established from single epiblast-like adult stem cells of embryonic stem cell characteristics by using CM of proliferating epiblast-like cells but standard growth media alone fail to produce clonal lines [7]. These results also suggest that CM of cultured cells can support the replication of stem cells which are otherwise unable to replicate. However, full potentiality CM has not yet been explored to use them as a supplement of bovine sera for regular culture of normal cells of limited replication. It is possible that normal cell-derived conditioned media are not used as a component of growth media for regular culture  of cells because of the difficulty of generating long-termgrowing cell lines from normal somatic cells without genetic manipulation. Also, adequate information on the stability of bioactive factors of CM after prolonged preservation is lacking. We have demonstrated recently that the CM of immortal hybrid cells retains biological activities at least for six months when preserved in the refrigerator and longer period if stored in the deep freeze [37]. The present study demonstrates that CM of highly proliferative normal ADSCs not only maintains self-renewal but also transforms other cells. Preservation of CM of proliferative normal cells and their subsequent use for improved proliferation of other cells could also reduce the expenses of regular cell culture, because presently CM of all types of cultured cells is discarded as waste. Importantly, culture of primary cells in the presence of CM not only prevents cellular senescence but produces large quantities adult stem cells with high proliferation which are useful for both research and therapeutic application.

The novel cell culture method described in the present study for the isolation and proliferation of ADSCs has many advantagescompared to conventional methods: (i) other than the use of culture medium containing fetal bovine serum this method does not require any special reagent and therefore any laboratory with basic tissue culture facilities can reproduce this method, (ii) this method allows the establishment of large quantity of cells from the excised adipose tissue in a shortest possible time by preventing cell loss from the culture, (iii) this method is economical because no medium change is needed until the culture flask becomes confluent with cells and at this stage cells are be detached by trypinization and passaged in new culture flasks with medium consisting of 25% CM and 75% FBS-containing fresh medium allowing improved cell proliferation and the remaining CM can be preserved for future use, (iv) CM of highly proliferating ADSCs can be used for morphological transformation of other type of cells. The new method is named as “conditioned media-mediated Cell Amplification, (cmCellAmp)”.

Future functional studies of the ADSCs isolated by the present method will reveal whether the popular notion of limited existence of somatic stem cells in the tissue is correct or not. In addition, functional analysis of morphologically transformed somatic cells derived from other tissues will reveal whether specific culture condition can convert normal somatic cells into multipotent stem cells or not. Both the questions are fundamentally important and answers of these questions have far-reaching implications both in basic research and in regenerative medicine.

Conclusions

Adipose-tissue derived stromal cells (ADSCs) are multipotent mesenchymal stem cells with the ability of differentiation into various cell types making it a useful source for cell replacement therapies. ADSC is commonly isolated from subcutaneous adipose tissue by collagenase digestion of biopsies followed by removal of undigested tissues and seeding of the dissociated cells onto plastic. This procedure allows the establishment of a tiny fraction of cells in culture. Development of new methods would be desirable to recover most cells of the biopsies which would reduce the time between biopsy collection and transplantation for quick replacement therapies. Present report describes a new cell culture method where almost all cells of subcutaneous fat tissue of horse can be established in culture by mincing the biopsies with a surgical scissor and culturing of fragmented tissue in RPMI 1640 with 15% fetal bovine serum for 2-3 weeks without change of medium. The conditioned media of proliferating ADSCs maintain self-renewal of fibroblast-like cells and morphologically transformed horse skin epidermal cells into fibroblast-like cells. The new method has direct application both in basic research and in regenerative medicine. The new method is named as “conditioned media- mediated Cell Amplification, (cmCellAmp)”.

Acknowledgements

This work was partly supported by Swedish Cancer Society through a grant to the author. The author gratefully acknowledges the contribution of Chris A. Sharp, Charles Salt Centre for Human Metabolism, Robert Jones & Agnes Hunt Orthopaedic & District Hospital NHS Trust, Gobowen, United Kingdom, for careful reading of the materials and method section of the manuscript.

Note: This method has been tested with rat skin, mouse skin and mouse whole embryo and found to produce similar results with horse skin culture

References

1.Thomson JA, Odorico JS. Human embryonic stem cell and embryonic germ cell lines. Trends Biotechnol. 2000, 18(2):53- 57.

2.Wobus AM. Potential of embryonic stem cells. Mol Aspects Med. 2001, 22(3):149-164.

3.Rippon HJ, Bishop AE. Embryonic stem cells. Cell Prolif. 2004, 37(1):23-34.

4.Czyz J, Wiese C, Rolletschek A, Blyszczuk P, Cross M, Wobus AM. Potential of embryonic and adult stem cells in vitro. Biol Chem. 2003, 384(10-11):1391-1409.

5.Forbes SJ, Vig P, Poulsom R, Wright NA, Alison MR. Adult stem cell plasticity:new pathways of tissue regeneration become visible. Clin Sci (Lond). 2002, 103(4):355-369.

6.Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell. 2004, 116(5):639-648.

7.Young HE, Duplaa C, Yost MJ, Henson NL, Floyd JA et al. Clonogenic analysis reveals reserve stem cells in postnatal mammals. II. Pluripotent epiblastic-like stem cells. Anat Rec A Discov Mol Cell Evol Biol. 2004, 277(1):178-203.

8.Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284(5411):143-147.

9.Sethe S, Scutt A, Stolzing A. Aging of mesenchymal stem cells. Ageing Res Rev. 2006, 5(1):91-116.

10.Bellantuono I, Keith WN. Stem cell ageing:does it happen and can we intervene? Expert Rev Mol Med. 2007, 9(31):1-20.

11.Giordano A, Galderisi U, Marino IR. From the laboratory bench to the patient’s bedside:an update on clinical trials with mesenchymal stem cells. J Cell Physiol. 2007, 211(1):27-35.

12.Da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci. 2006, 119(Pt 11):2204-2213.

13.Jackson L, Jones DR, Scotting P, Sottile V. Adult mesenchymal stem cells:differentiation potential and therapeutic applications. J Postgrad Med. 2007, 53(2):121-127.

14.Barry FP, Murphy JM. Mesenchymal stem cells:clinical applications and biological characterization. Int J Biochem Cell Biol. 2004, 36(4):568-584.

15.Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells:revisiting history, concepts, and assays. Cell Stem Cell. 2008, 2(4):313-319.

16.Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum. 2005, 52(8):2521-2529.

17.Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol. 2005, 33(11):1402-1416.

18.Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H et al. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res. 2007, 327(3):449-462.

19.Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006, 24(5):1294-301.

20.Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW et al. Multilineage cells from human adipose tissue:implications for cellbased therapies. Tissue Eng. 2001, 7(2):211-228.

21.Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002, 13(12):4279-4295.

22.Fraser JK, Wulur I, Alfonso Z, Hedrick MH. Fat tissue:an underappreciated source of stem cells for biotechnology. Trends Biotechnol. 2006, 24(4):150-154.

23.Schäffler A, Büchler C. Concise review:adipose tissue-derived stromal cells–basic and clinical implications for novel cell-based therapies. Stem Cells. 2007, 25(4):818-827.

24.Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007, 100(9):1249-1260.

25.Strem BM, Hicok KC, Zhu M, Wulur I, Alfonso Z et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med. 2005, 54(3):132-141.

26.Nakagami H, Morishita R, Maeda K, Kikuchi Y, Ogihara T et al. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb. 2006, 13(2):77-81.

27.Lee RH, Kim B, Choi I, Kim H, Choi HS et al. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem. 2004, 14(4-6):311-324.

28.Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM et al. Characterization of equine adipose tissue-derived stromal cells:adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells. Vet Surg. 2007, 36(7):613-622.

29.Zuk PA. The adipose-derived stem cell:looking back and looking ahead. Mol Biol Cell. 2010, 21(11):1783-1787.

30.Helder MN, Knippenberg M, Klein-Nulend J, Wuisman PI. Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue Eng. 2007, 13(8):1799-1808.

31.Mizuno H. Adipose-derived stem cells for tissue repair and regeneration:ten years of research and a literature review. J Nippon Med Sch. 2009, 76(2):56-66.

32.Mohamed-Ali V, Pinkney JH, Coppack SW. Adipose tissue as an endocrine and paracrine organ. Int J Obes Relat Metab Disord. 1998, 22(12):1145-1158.

33.Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab. 2004, 89(6):2548-2556.

34.Fonseca-Alaniz MH, Takada J, Alonso-Vale MI, Lima FB. Adipose tissue as an endocrine organ:from theory to practice. J Pediatr (Rio J). 2007, 83(5 Suppl):S192-S203.

35.Islam MQ, Panduri V, Islam K. Generation of somatic cell hybrids for the production of biologically active factors that stimulate proliferation of other cells. Cell Prolif. 2007, 40(1):91- 105.

36.Islam MQ. Improved proliferation of normal somatic cells by treatment of conditioned media derived from hybrid cells. In: Progress in cell growth process research, Edited by Takumi Hayashi, New York: Nova Science Publishers, Inc; 2008:89-118.

37.Islam MQ, Islam K, Sharp CA. Epigenetic reprogramming of nonreplicating somatic cells for long-term proliferation by temporary cell-cell contact. Stem Cells Dev. 2007, 16(2):253- 268.

38.Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol. 2000, 227(2):271-278.

39.Kouichi Hasegawa, Tsuyoshi Fujioka, Yukio Nakamura, Norio Nakatsuji, Hirofumi Suemori. A Method for the Selection of Human Embryonic Stem Cell Sublines with High Replating Efficiency after Single-cell Dissociation. Stem Cells. 2006, 24(12):2649–2660.

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