Patent Application: US-73050410-A

Abstract:
the present invention provides platelet production methods . the method comprises the steps of providing a cellular material and culturing the cellular material , wherein platelets are produced . the culturing may be performed on 2d or 3d cell support structure or in suspension culture . in some embodiments , all or a part of the 2d or 3d culturing may be performed in a bioreactor . in some embodiments , the method may further comprise a step of isolating a subset of cells from the starting cellular material , wherein the isolated subset of cells is then cultured , wherein platelets are produced . in yet other embodiments , the method comprises the steps of providing a cellular material , isolating a subset of cells , seeding the subset of cells into a 3d scaffold , culturing the subset of cells in a 3d scaffold , seeding the cultured subset of cells into a bioreactor , culturing the subset of cells in a bioreactor , and harvesting the cells from the bioreactor , wherein platelets are produced .

Description:
the present invention will now be described by reference to some more detailed embodiments , with occasional reference to the accompanying drawings . this invention may , however , be embodied in different forms and should not be construed as limited to the embodiments set forth herein . rather , these embodiments are provided so that this disclosure will be thorough and complete , and will fully convey the scope of the invention to those skilled in the art . unless otherwise defined , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . the terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention . as used in the description of the invention , the singular forms “ a ,” “ an ,” and “ the ” are intended to include the plural forms as well , unless the context clearly indicates otherwise . all publications , patent applications , patents , and other references mentioned herein are incorporated by reference in their entirety . unless otherwise indicated , all numbers expressing quantities of ingredients , reaction conditions , and so forth used in the specification and claims are to be understood as being modified in all instances by the term “ about .” accordingly , unless indicated to the contrary , the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention . at the very least , and not as an attempt to limit the application of the doctrine of equivalents , each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches . notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations , the numerical values set forth in the specific examples are reported as precisely as possible . any numerical value , however , inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements . every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range , as if such narrower numerical ranges were all expressly written herein . cord blood is used clinically as a source of hematopoietic progenitors . it is readily available and largely merely discarded after delivery . on the other hand , there are only a limited number of cells available in each collection . disclosed herein is use of cord blood as a source of cells for production of platelets in vitro . it has been shown that growth in a 3d matrix of nonwoven polyester fabric enhances expansion of committed progenitors , compared to growth on a 2d surface [ 19 ]. in addition to using fibrous polyester 3d scaffolds described herein , use of purpose - built hydrogel scaffolds is disclosed . colloidal crystals can be self - assembled by sedimentation / evaporation of corresponding dispersions and then annealed to form solids [ 20 - 23 ]. inverted colloidal crystals ( icc ) have unique advantages as cell culture substrates , including unprecedented level of control over the 3d geometry of cellular matrixes ; high porosity ( 74 % void volume ); exceptionally high interconnectivity — each cavity has a total of 12 neighboring ones with 12 interconnecting channels ; spheroid shape of the cavities hosting cells , stimulating intercellular interactions ; and simplicity of preparation . icc scaffolds can be made without any highly specialized equipment . demonstrated herein is that utilization of this geometry constructed with biocompatible hydrogel provides a useful milieu for marrow cell growth and platelet production . a number of bioreactor types have been used in hematopoietic stem cell expansion and research , including stirred tank suspension , fluidized bed , fixed bed , airlift , perfusion chamber , and hollow fiber [ 26 ]. the most frequently described , growth in ( 2d ) static flasks or suspension , limit long term cell - cell or cell - matrix interactions . to see if a more physiologic milieu would further promote platelet production , we utilized woven polyester surgical fabric scaffold in a 3d single - pass perfusion bioreactor system . the bioreactor module is designed to allow maturing cells to settle continuously into the spent medium while immobilized parent cells are out of the direct medium flow path ( fig1 a ). the system is modular , and we have successfully used multiple bioreactor modules in parallel to facilitate cell production . this may avoid problems like those described by matsunaga et al , in which they needed a very low concentration of early progenitors to produce large numbers of platelets in their feeder layer system [ 1 ]. our system provides a large effective surface area , both as a result of the use of a 3d fabric scaffold and of the modular nature of the system . umbilical cord blood units were obtained from normal full - term deliveries after institutional review board approval and informed consent . light density cells were isolated from citrated cord blood using discontinuous density centrifugation using ficoll - paque plus ( ge healthcare biosciences , uppsala , sweden ). cd34 - positive selection was conducted using a macs direct cd34 progenitor cell isolation kit ( miltenyi biotec , anaheim , calif .). in our laboratory this historically produces greater than 90 % cd34 positive cells , as confirmed by flow cytometry . only samples with greater than 95 % viability as determined by trypan blue dye exclusion were used in further studies . two types of scaffolds were used . the first was 1 . 9 cm diameter × 1 mm depth disks of sterile surgical grade bard polyester velour woven fabric ( c . r . bard , inc ., humacao , puerto rico ). this was used in both 12 well plates and the perfusion bioreactor system . the second scaffold type was constructed from hydrogel . for manufacture of these scaffolds , colloidal crystal ( cc ) was used as a template for the 3d poly ( acrylamide ( am )) hydrogel cell culture scaffold . the fabrication protocol of the cc resembles the process developed by cuddihy and kotov , and its transformation to inverted colloidal crystal ( icc ) hydrogel scaffold resembles the process developed by lee et al [ 21 , 27 ]. the cc was constructed by sedimentation of soda lime glass spheres while providing agitation via sonication . to further ensure a high degree of orderliness , the sedimentation rate was retarded by focusing the spheres into a narrow channel prior to entering the mold and by the use of ethylene glycol ( sigma , st louis , mo .) as the sedimentation medium . after the thickness of the cc grew to a desired height , the cc was dried at 160 ° c . and annealed at 700 ° c . for 4 hours . the heat treatment caused partial melting at the surfaces , which resulted in annealing of spheres with their adjacent neighbors . upon the fabrication of colloidal crystal , the poly ( am ) hydrogel precursor , composed of a 30 wt % acrylamide ( sigma ) precursor containing 5 wt % of n , n - methylenebisacrylamide ( nmba ) cross - linker , was infiltrated into the cc via centrifugation . low viscosity of the precursor solution ensured complete infiltration in between the beads . polymerization was initiated by adding aqueous potassium persulfate ( kps ) solution ( 1 w / v %) at a ratio of 1 : 10 by volume in an oxygen - free environment . after polymerization , excess hydrogel pieces were removed and the hydrogel encapsulated cc was then immersed in a hydrofluoric acid ( hf ) and subsequent hydrochloric acid ( hcl ) bath to extract the internal glass spheres , resulting in a disc - shaped 3d icc poly ( am ) hydrogel scaffold . the icc poly ( am ) hydrogel scaffold was detoxified of the etchants by excessive rinsing with ph 10 buffer , 0 . 1 m calcium chloride solution and water followed by freeze drying . the surfaces of icc hydrogel scaffolds were modified through layer - by - layer deposition of positively charged 0 . 5 wt % poly ( diallyldimethylammonium chloride ) ( pdda , mw = 200 , 000 , sigma ) solution and negatively charged 0 . 5 wt % clay platelet ( average 1 nm thick and 70 - 150 nm in diameter , southern clay products ) dispersion with deionized water rinse in between the steps . duration of each deposition and rinse was 15 minutes and a cycle of pdda adsorption / rinse / clay adsorption / rinse process was repeated 5 times . following the deposition of pdda , selected scaffolds were coated separately with using 1 mg / ml fibronectin in phosphate buffered saline ( pbs ) or 10 ug / ml tpo in pbs for 1 hour at room temperature , then washed in pbs to remove unbound protein . the hydrogel scaffolds , 9 to 11 mm diameter disks approximately 1 mm thick , were used in well plates . cavities in the hydrogel were 355 to 420 um in diameter , and they interconnected with adjacent cavities by openings that averaged 10 to 15 % of the cavity diameter . they were placed on sterile polyester 1 mm mesh ( textile development associates , franklin square , n . y .) over a conical funnel to facilitate seeding of cells into the hydrogel scaffolds by gravity filtration of cell - containing medium through the scaffold . after seeding , hydrogel scaffolds were placed in 24 - well tissue culture plates and maintained in 1 ml medium at 37 ° c . humidified air 5 % co 2 . the perfusion bioreactor modules are self - contained cell support systems that facilitate medium and gas exchange over and under a cell support scaffold ( fig1 a ). the device is constructed from polycarbonate . the gas - permeable membrane is made from smooth finish fluorinated ethylene propylene ( mcmaster carr , aurora , ohio ). three disks of woven polyester fabric , 1 . 9 cm diameter and 1 mm thick , used as cell scaffolds , were fixed in the center . medium flows over and under the disks , but not through them , minimizing shear forces . non - adherent cells produced during incubation fell into the lower medium space , from which they were be collected in the discard medium pushed out of the bioreactor module when fresh medium is added . for gas exchange , the medium is separated by a sterile smooth - finish fluorinated ethylene propylene 0 . 025 mm thick gas - permeable membrane ( mcmaster carr aurora , ohio ) barrier from a continuous flow of humidified 5 % co 2 in air through the bioreactor module . each bioreactor module has separate seeding and sampling ports allowing each module to be manipulated or removed independently of the other modules despite connections through the tubing network . in the experiments described , each module supported three bard fabric cell scaffold disks . bioreactor modules were run in parallel , with parallel bioreactor modules connected to a single fresh medium source . to seed the cells into the modules , cell - containing medium was injected via a luer lock connection . the tubing leading to and from the bioreactor module was clamped to create a cross flow of medium across the cell chamber containing the pre - placed fabric scaffold . the cells were then injected into the bioreactor module from the syringe such that the medium flow was through the matrix from top to bottom , resulting in trapping of cells within the scaffold . cells were then permitted to adhere to the scaffold for 24 - 72 hours before the tubing clamps were removed and regular cross flow of medium resumed . medium exchange and cell harvests were accomplished simultaneously in the single pass bioreactor module . daily , medium ( 3 ml ) containing non - adherent cells were withdrawn from the bioreactor as fresh medium entered the module from the medium supply . for platelet function studies , a syringe containing 35 ml of glucose - based platelet storage medium , as previously described by holme et al , was attached to the harvest port of the bioreactor module [ 28 ]. three ml of platelet differentiation medium containing shed cells was withdrawn into the syringe . cd34 - enriched cells were initially expanded in liquid culture at 37 c humidified air mixed with 5 % co 2 for 48 - 72 hours in iscove &# 39 ; s modified dulbecco &# 39 ; s medium ( imdm ; gibco , invitrogen , carlsbad , calif .) expansion medium containing 10 % fetal bovine serum ( fcs ; jrh bio sciences lenexa , kans . ), 50 ng / ml scf , 50 ng / ml fl , 10 ng / ml tpo , and 10 ng / ml il - 6 . all growth factors were from r and d systems , minneapolis , minn . after the liquid culture expanded cells were seeded separately into each type of support scaffold . the cells were maintained for 7 days with daily medium changes after 48 hours in hematopoietic progenitor maintenance medium containing imdm , 10 % fcs , 10 % horse serum ( jrh bio sciences ), 0 . 25 um hydrocortisone , 10 ng / ml tpo , and 25 ng / ml fl . all additional medium additives and growth factors were from r and d systems , minneapolis . after 7 days , the medium was changed to differentiation medium to promote megakaryocyte and platelet production ( src differentiation medium ) that consisted of imdm , 10 % fbs , 30 ng / ml tpo , 1 ng / ml scf , 7 . 5 ng / ml il - 6 , 13 . 5 ng / ml il - 9 , and 2 . 5 um su6656 ( sigma ), a src kinase inhibitor . this regime was used to compare incubation in tissue culture wells , in polyester fibrous scaffolds in wells , and in polyester fibrous scaffolds in the bioreactor system . each experiment was performed using a unit of cord blood ; approximately two million cd34 positively - selected cells were used for each of the three conditions . one aliquot was cultured at the bottom of 6 tissue culture wells in a 12 well plate , one was cultured in six 1 . 9 cm diameter × 1 mm woven polyester scaffold maintained in 6 tissue culture wells in a 12 well plate , and the last was cultured in the 3d single pass perfusion bioreactor , using two modules , containing a total of 6 woven polyester scaffolds , connected in parallel to gas and medium supplies , and maintained at 37 ° c . to determine the possible effect of increasing il - 6 and adding il - 11 , one experiment was performed using imdm containing 10 % fbs , 1 % bovine serum albumen , 25 ng / ml tpo , 25 ng / ml il - 3 , 50 ng / ml il - 6 , and 10 ng / ml il - 11 as differentiation medium ( il - 6 ,- 11 medium ). one experiment to compare effects of different protein coatings was performed in hydrogel scaffolds in tissue culture wells using src differentiation medium . then , to determine the possible effect of more il - 6 and added il - 11 in the protein - coated hydrogel scaffolds , another experiment using platelet differentiation medium containing il - 11 and a higher dose of il - 6 was performed . src differentiation medium was used from day 7 to 14 , when the differentiation medium was switched to il - 6 ,- 11 medium on day 14 . light microscopy was performed on cells harvested from culture conditions on an axioskop 2 zeiss microscope ( carl zeiss , thornwood , n . y .) utilizing the axiocam imaging system . wright &# 39 ; s - stained cytospin ( shandon , pittsburgh , pa .) preparations were examined for morphology . for fluorescent studies of hydrogel scaffold sections , wedges were cut from whole scaffolds on day 32 following cell seeding . the hydrogel scaffold sections were incubated with fitc - labeled anti - cd41 or control igg fitc antibodies , washed in pbs and placed on glass slides under coverwell imaging chamber gaskets ( molecular probes , eugene oreg .). for transmission electron microscopy ( tem ), an enriched platelet suspension was fixed in a 10 : 1 solution of phosphate buffered saline and 2 . 5 % glutaraldehyde , ph 7 . 4 . following fixation , the platelet suspension was transferred to 2 % liquefied agarose at 45 ° c . the agarose block containing the visible platelet pellet was then processed for tem following the methods detailed by mclean et al with the exception that for fixation , an epon - ethanol mixture was used instead of spurr [ 29 ]. studies were performed using a fei technai g2 spirit transmission electron microscope ( eindhoven , netherlands ). cells for flow cytometric analysis were washed in ca ++/ mg ++ free dulbecco &# 39 ; s phosphate buffered saline to remove culture medium and labeled with fitc / pe conjugated antibodies ( immunotech , marseille , france ) for surface antigens , including those for cd34 , cd41 , cd62 , and cd63 . excess antibody was removed by washing , and the fluorescent cell analysis was performed on a bd facs calibur system flow cytometer ( bd biosciences , san jose , calif .). for comparison neonatal platelets were isolated from citrated cord blood by 120 × g centrifugation for 15 minutes at room temperature . neonatal platelets were then washed and labeled as described for experimentally - produced platelets . to determine the functional properties of platelets harvested from 2d , 3d , and 3d perfusion bioreactor growth conditions , as well as hydrogel scaffolds , harvested platelets were collected , washed , and resuspended in 35 ml glucose storage buffer , incubated with or without 1 . 0 u / ml thrombin for 10 min at 37 ° c ., and observed for aggregation using phase microscopy [ 28 ]. to detect platelet activation antigens cd62 and cd63 , platelets were isolated and enriched as previously described . the platelet - rich portion was then resuspended in ca ++/ mg ++ free dulbecco &# 39 ; s pbs and incubated with or without 1 . 0 u / ml thrombin for 10 min at 37 ° c . platelet activation surface antigens cd62 and cd63 were measured by flow cytometry before and after exposure to thrombin . cd34 positively - selected cord cells were expanded for 48 to 72 hours in liquid culture . this increased the number of cells from about 2 million to about 15 million , and increased the number of cd34 + cells 4 to 5 fold . following this , cd34 positively - selected cord cells from the same donor were divided in equal numbers among three conditions . cells were further incubated in wells ( 2d ), introduced into fabric scaffolds ( 3d ), or infused into perfusion bioreactor modules containing identical scaffolds ( 3d bioreactor ; table 1 ). daily , old medium was removed and fresh medium was added . cells in the old medium from each condition were harvested by centrifugation , counted , and further characterized . we connected two identical bioreactor modules in parallel for these experiments . at 7 days , growth factors were changed to il - 6 , il - 9 , scf , tpo , and su6656 , a src kinase inhibitor . we used a modification of the method described by gandhi et at for 2d culture [ 11 ]. we replaced il - 3 with il - 9 based on the work of cortin et al , who carefully compared a large number of cytokine combinations for their megakaryocyte and platelet production potential , and that of fujiki et at [ 30 , 31 ]. in three identical experiments , in tissue culture wells ( 2d ), 2 . 3 , 1 . 8 , and 1 . 7 × 10 6 morphologic platelets were produced from day 12 to 25 ; in 3d scaffolds in wells ( with a total of 6 disks per experiment ), 10 , 6 . 9 , and 9 . 3 × 10 6 from day 12 to 37 ; and in 3d scaffolds in the modular perfusion bioreactor , 31 , 31 , and 36 × 10 6 from day 8 to day 40 ( fig1 b ). when normalized to number of platelets produced per cd34 positively - selected expanded cord blood cell , the 3d bioreactor produced statistically significantly higher yield per starting cell compared to either 2d or 3d production in wells . when we increased the dose of il - 6 and added il - 11 we found a considerable increase in length of platelet production and numbers produced daily ( table 1 and fig1 d ). for this experiment we used 6 million cd34 positively - selected cord cells that had been expanded in brief liquid culture , putting 2 million cells in each of three bioreactor modules ( table 1 ). even considering the increased starting numbers over the previous experiments , the number of platelets produced per day was increased dramatically over the previously - used cytokine combination . approximately twice the number of putative platelets per expanded cd34 positively - selected cell that we observed with src differentiation medium was produced . compound microscopy of wright - stained smears and cytospins showed a heterogeneous mixture of normal and atypically - shaped and - sized platelets ( fig1 e ). tems showed the presence of alpha and dense granules , mitochondria , open cannicular elements , and circumferential microtubules similar to that seen with concurrent fresh cord blood platelets , although there were somewhat more microparticles , ghosts , and abnormally - shaped and - sized platelets , with some degranulation . serial flow cytometry in one experiment showed decreasing contamination of cd41 negative particles as cd41 positive small particles increased ( fig1 c ). the day 7 results for the platelet differentiation medium and the control medium were identical , as expected . at 14 days , a large population of small ( low forward scatter ) cd41 positive particles were recorded in the sample from the differentiation bioreactor ; these were presumably platelets . there was also a significant population of small particles that were not cd41 positive , probably cellular debris . this debris almost disappeared at the day 21 and day 28 time points , while the small - sized cd41 positive population persisted . the platelets collected aggregated in response to thrombin , as did neonatal control platelets . we analyzed the platelets for cd62 and cd 63 expression before and after thrombin exposure , and compared this to neonatal platelets harvested from cord blood within 24 hours of delivery , using flow cytometry ( fig1 f ). the bioreactor - produced platelets showed considerable cd62 and cd63 activation above that seen with cord blood platelets . thrombin activation increased the expression of both markers of platelet activation above the baseline expression . to see the potential effect of embedded proteins in 3d , we used hydrogel scaffolds coated with clay without embedded protein or decorated with fibronectin and / or thrombopoietin , in tissue culture wells ( fig2 and table 1b ). the results suggested fibronectin and tpo increased the amount and duration of platelet production , over plain clay coating alone ( fig2 b ). increasing il - 6 and adding il - 11 increased platelet production , and the effect of protein coatings was largely lost ( fig2 b ). total production normalized to starting expanded cd34 positively - selected cells approximately doubled ( table 1 ). flow / cross flow in the bioreactor system : we postulated that instituting continuous flow during incubation in the bioreactor would increase platelet production . we based this expectation on descriptions in the literature of up to a 20 fold increase in platelet production in a 2d flow system . [ 47 , 45 ] we compared flow across the cell - scaffold construct achieved by blocking the lower medium input port and the upper output port with flow parallel to the cell - scaffold construct ( all ports open ). a 3 ml bolus was used for harvest in both the cross and continuous flow modes , and for the intermittent flow bolus alone method used previously . we found a marked increase in platelet production , up to a 3 - fold increase from day 17 onward , with cross - flow but not parallel flow ( fig4 ). we explored decreased oxygen level during culture to enhance platelet production . our reasoning was that there is a lower level of oxygen in the marrow space near the surrounding bone where early hematopoietic cells are found . we compared 20 % o 2 vs . 5 % o 2 . we found that if the entire platelet production scheme outlined above was performed at 5 % o 2 , there was a marked diminution in platelet production . on the other hand , if the initial 2 - 3 day expansion in liquid culture and the 7 day expansion in the 3d scaffold were performed at 5 % and the differentiation stage was carried out at 20 % o 2 , we found an approximate doubling of the number of platelets produced in the 3d and 3d bioreactor systems . in the disclosure presented here , we avoided the use of feeder cells . some aspects of the 3d niche microenvironment can be supplied in a culture system in which a feeder cell layer is used . this feeder layer , often cell lines or mscs , may supply points of contact for adherent cells , molecules that interact with receptors on the target cell &# 39 ; s surface , or the opposite , and may also produce soluble chemokines or cytokines necessary for the target cells growth and survival [ 15 - 17 ]. osiris therapeutics , inc ., has reported successfully growing marrow cd34 positive cells on mscs , with formation of megakaryocyte colonies in serum - free medium without added cytokines [ 18 ]. because of the difficulty of obtaining and maintaining a feeder layer in a large scale system , we sought , successfully , to determine whether we could produce functional platelets using a feeder - free 3d culture system . we demonstrated marked improvement in platelet production when 3d cell - scaffold constructs were created and grown in a 3d modular single - pass perfusion bioreactor system . several types of bioreactors have been described for growth of hematopoietic cells . the most simple , suspension or 2d adherent - cell cultures , allow minimum cell - cell contact . most flow systems such as packed bed reactors produce significant shear forces on the growing cells [ 32 - 34 ]. the aastrom replicell , initially introduced to expand marrow - derived progenitors , uses a single pass 2d system [ 35 ]. other 3 - dimensional culture methods for human cells that utilize some form of bioreactor include a tantalum - coated porous biomaterial ( cytomatrix ) to culture and expand hematopoietic progenitor cells from bone marrow for up to 6 weeks and umbilical cord blood cd34 + cells for up to 2 weeks [ 36 , 37 ]. banu et al , successfully cultured cd34 + cells from human bone marrow on a porous three - dimensional biomatrix ( cellfoam ™) for up to 6 weeks [ 38 ]. zhao and ma reported the used pet matrix scaffolds in a bioreactor device to culture human mesenchymal stem cells ( mscs ) for a period of 40 days [ 39 ]. braccini et al , also reported expansion of mscs in a 3d scaffold - based bioreactor system , in addition to co - culture of hematopoietic progenitor cells [ 40 ]. our bioreactor system is purpose - built for hematopoietic culture ; it allows continuous collection of non - adherent cells . at the same time , it allows independent control of medium and gas flow , as well as a variety of medium utilization methods . the system as described here uses a discontinuous ( every 24 h ) single - pass scheme , but it can be configured for continuous or pulsed flow with or without automatic recycling of the output medium . the environment engendered by the bioreactor module , even with the limited ( once per 24 hours ) flow , allows nutrient , waste , and gas exchange above and below the 3d scaffold ; we believe this accounts for the greatly increased platelet production in the bioreactor system . to test function , we performed aggregation studies using a method for small numbers of platelets [ 28 ]. there was also a measurable response to thrombin using antibodies to cd62 and cd63 by flow cytometry . the flow studies , while showing an increment in activation with thrombin , also showed that these platelets were activated in the absence of added thrombin . several possible explanations , including activation on contact with portions of the bioreactor system flow path or activation by the cytokine - containing differentiation medium , are possible . we also noted somewhat deranged morphology compared to similarly - prepared neonatal platelets in both wright &# 39 ; s stained preparations and with tem . we think that the activation sans added agonists and the abnormal morphology can be remedied by several future changes in the system , notably by using continuous medium flow into the bioreactor modules with resulting continuous collection of shed platelets , and by modifying the surfaces of the bioreactor system and scaffolds . to test platelet formation in the presence or absence of adherent tpo or fibronectin . we used poly ( am ) hydrogel as the substrate material for an inverted crystalloid scaffold , chosen for its biocompatibility , mechanical strength and transparency . mechanical rigidity resulted in a firm icc structure and transparency allowed facile optical analysis . we modified the hydrogel surface using by layer - by - layer deposition of positively charged 0 . 5 wt % pdda solution and negatively charged 0 . 5 wt % clay platelet dispersion [ 43 ]. the lbl deposition of nano - structured clay platelet and pdda increased nano - scale roughness and created surface charge and stiff film , which worked in concert , along with adhered proteins , to bind and stimulate cells . we found that with the combination of both tpo and fibronectin , utilizing the src kinase inhibitor medium , platelet production lasted longer at a higher level vs . only one or the other or neither . on the other hand , when we maximized platelet production using il - 6 and il - 11 , little increase was seen in platelet numbers produced with either or both coating proteins . our hypothesis is that the tpo has both a stimulating and a binding effect in our system , and that fibronectin also binds both progenitors and megakaryocytes during hematopoiesis in the system . the need for the bound proteins to increase platelet production is overcome by increasing fluid - phase il - 6 and adding il - 11 . the current results show continuous prolonged production of platelets using a 3d single - pass intermittent - flow perfusion bioreactor system , markedly more than with conventional 2d culture . while we expected to produce platelets with the system , its ability to produce them over a long period of time ( several weeks ) was unexpected . it appears that the 3d milieu engendered by the scaffolds we have used , especially when used with our modular bioreactor system , allows asynchronous production of platelet progenitors and precursors for prolonged periods . even so , the number of platelets produced falls far short of the number needed for transfusion . future improvements to increase yield include adoption of methods to increase the number of early progenitors from cord blood ; longer periods of continuous perfusion , with or without medium recirculation ; and alterations in scaffold surface makeup and design . more than two million platelet transfusions are given in the us annually . the brief storage time of platelets , 5 to 7 days , often creates shortages in times of emergency and environmental crisis . these and other difficulties could be overcome with an in vitro platelet production system . the work presented here may provide a foundation for future development of such a clinical production system , in addition to creating an in vitro model of megakaryocytopoiesis and thrombopoiesis that can be used to study these processes and the effect of drugs and disease . yao c l ci , hsieh t b , hwang s m . a systematic strategy to optimize ex vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells . exp hematol . 2004 ; 32 : 720 - 727 . shim m h ha , blake n , drachman j g , reems j a . gene expression profile of primary human cd34 + cd3810 cells differentiating along the megakaryocyte lineage . exp hematol . 2004 ; 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