The Director of the United States Patent and Trademark Office Has received an application for a patent for a new and usefil invention. The title and descrip- tion of the invention are enclosed. The require- ments of law have been complied with, and it has been determined that a patent on the in- vention shall be granted under the law. Therefore, this United States Patent Grants to the person(s) having title to this patent the right to exclude others from making, using, offering for sale, or selling the invention throughout the United States of America or im- porting the invention into the United States of America for the term set forth below, subject to the payment of maintenance fees as provided by law. If this application was filed prior to June 8, 1995, the term of this patent is the longer of seventeen years from the date of grant of this patent or twenty years from the earliest effec- tive US. filing date of the application, subject to any statutory extension. If this application was filed on or after June 8, 11995, the term of this patent is twenty years from the US. filing date, subject to any statutory ex- tension. If the application contains a specific reference to an earlier filed application or ap- plications under 35 US.C. 120, 121 or 365(c), the term of the patent is twenty years from the date on which the earliest application was filed, subject to any statutory extensions. bs WE ste Direcor ofthe Unt States Patent a Tenant Ofce| i | ' eat ay United States Patent ‘Chu et al. 1US007323190B2 US 7,323,190 B2 *Jan. 29, 2008 (10) Patent No. (45) Date of Patent: (54) CELL DELIVERY SYSTEM COMPRISING A FIBROUS MATRIX AND CELLS (75) Inventors: Benjamin Chu, Setauket, NY (US); Benjamin S. Hsiao, Sotzuket, NY (US); tael Hadjiargyrou, Coram, NY (US); Dufei Fang, Painted Post, NY (US); Xinhua Zong, Centereach, NY (US); Kwangsoke Kim, Setauket, NY (us) (73) Assignee: "The Research Foundation at State University of New York, Stony Brook, NY Us) (#) Notice: Subject to any disclaimer, the term ofthis patent is extended or adjusted under 35, USC. 154(b) by 416 days ‘This patent is subject to a terminal dis- cline, 21) Appl. No 10919,616 (22) Filed: Aug. 17, 2004 5) UUs 200510014252 Al Jan. 20, 2005, Related US. Application Data (63) Continuation of application No, 09953411, filed on Sep. 14, 2001, now Pat. No. 6,790,455. () Ina. AGIE 2100 (2006.01) crn 1108 (2006.01) Cran 1104 (2006.01) CRIN 106, (2006.01) CRN 508 (2006.01) (2) US.cL 424/426; 424093,7; 435/180; 435/182; 435/395 (58) Field of Classification Search None See application fle for complete search history. (56) References Cited US. PATENT DOCUMENTS 3975565 A ¥11976 Kendall aaa 3043331 A W977 Main "anise 4323525 A 4/1982 oma 26424 345414 A W/19R2 Boma et savas 4468902 A 911986 MeCrady ea ‘sao (663286 A 5/1987 “Tang et “ssii78 $469,186 A W987 Borat “264/16 4803168 A 211980 unis, Je «43524022 4310180 A 1989 ner 4251788 4878908 A 117989 Matinee SOB AS11867 A 31980 Bure eta 6422 3066755 A 11/1991 Lenses ‘608481 SIGTIT A 511982 Moo Young eta. ran 435/178 5296172 A 31998 Davis ea 26424 SS67612 A 10/1956 Vacant etal 135200.23, S569.508 A 10/1995 Van der Loo ef a. 4247298 S783111 A T998 Thala et 252/500, 6031 A 12000 ‘euon 6081148 A 272000 ‘vil 6090910 A 772000 Osawa 6106913 A 872000. Scardino et a 44363 671610 BL 12001 Vacant eta “24106 177095 BL 12001 Sawhney eta. aus {6206914 BL 32001 Soykan eal nro 62142 621881 BI 472001 Meluch eal S210 6231881 BI 52001 Usala eta. ‘ewias 6238,705 BI 572001 Live a 24501 6.685956 B2* 22004 Chu oa, aus 6589,374 B2* 272004 Chu ot ua 699055 B2* 972004 Chu eta ouina TAMT65 82* 22007 Chu ota ues FOREIGN PATENT DOCUMENTS wo woos? 1998 Wo WOOIR6610 AL 472001 Wo WOOU/ZT365 Ai 472001 (OTHER PUBLICATIONS Bezwada otal, “Poly(p-Dionanons) and ls Copolymers,” Hand: book of Biodegradable Polymers, 29-61 (1997) Denis eta, Polymer Hybrid Nano/Micro Composites," Proceed- ings of the American Society for Composites Nath Techneal Con- ference, pp. 657-65 (199). * cited by examiner Primary Examiner—David M. Naft (14) Atorney, Agent, or Firm—HolTman & Baron, LLP on ABSTRACT Cell storage and delivery systems and methods for storing and delivering viable cells to a mammal are disclosed. The ‘ell storage and delivery systems include a biodegradsble and/or bioabsorbable fibrous matrix physically associated with viable cells to contain and release the cells at a controlled rate. The biodegradable and/or bioabsorable mairix can be formed by electrospinning fibers of biode- gradable and/or bioabsorbable fiberizable material. The methods include methods for storing viable cells and for delivering viable cells to a mammal sing the cel storage and delivery system, 15 Claims, 12 Drawing SheetsUS 7,323,190 B2 25 Example 2 ‘The cell release by the delivery system from Example 1 ‘was tested as follows: the pets dish containing the delivery system was brought to room temperature and $ mi of the ‘minimum esseaial medium solution, ineluding Fetal Bovine ‘Serum, was added into the petri dish containing the layered cellmembrane composite. The dish was maintained in an incubator under the following conditions: the temperature ‘was 37°C. in fully-humiditied atmosphere having 5% CO, in air, Several cell/membrane composites were used for the study. Individual composites were removed from the ineu- Dalor at diferent times for cell culture testing using an ‘optical microscopy technique. The number of cells was determined and recorded. ‘The typical cell release and ‘growth mechanism, which was observed visually, is shown schematically in FIG. 8. Referring to FIG. 8, the embedded cells $0 were found t0 bbe able to survive because there were many small pores on the top membrane layer 51 to allow transfer of oxygen and the nutrients tothe cells 52.'The top membrane $1 was used ‘only 28 mechanical restraining means without damaging the properties of cells. In the chosen stad, the cells started to be ‘leased from the membrane 83 in the first day. The released cells 54 would sic to the botiom of the petri dish $5, and the shape of cell was found to change from round to Nat 56, After attachment, the cells grew through proliferation, ‘The numbers of cells attached to the petsi dish were measured by an optical technique. The results are shown raphically in FIG. 9. A review of FIG. 9 reveals that more than 20% of the cells in the membrane were released in 2 days, and the released cells grew continuously. After | wesk, the cell numbers were found to increase 7 times compared to the number of cells in the initial condition. The optical microscopic photographs of the cells released from the membrane asa funedon of me also indicate that the PLA | membrane has not damaged the cell growth process Optical microscopic photographs, taken over time, are own in FIGS. 10()-10(). The photographs were taken at he following time intervals following the start of inca. “ion: FIG. 102)-2 days; FIG. 10()-1 week; FIG. 10()-2 “vecks; and FIG. 10(4)-6 weeks. A review of FIGS. 10(2- lish was covered with cells in 1 week, The calls continued i show a more dense population and finally formed a ineralised matrix after 6 weeks, Example 3 ‘The mechanical properties of an electrospun membrane ful asa carrer ina cell delivery and storage system was valuated as follows: A PLGA (75/25 PLA:PGA) membrane produce by a process similar to Example 1. The mibrane had a thickness in the range of about 150 nicrons. The membrane was cut into a 2,552.5 em strip and aluated fr flexibility by holding one end ofthe stip using ‘and twisting the strip. ‘The strip was then quenched in liguid nitrogen for 20 inves and then removed from the liguid nitrogen, The fozen strip was again evaluated for flexibility. FIG. 11 Mustrtes the Nexbiity before and after quenching. The Pembrane retained significant lexibility and never became ile or developed cracks when \wisied in a frozen sate. he full lexbility ofthe membrane returned upon reaching temperature, 26 Example 4 ‘The cell release by a eal delivery system having an electospan PLOAMactide mauix carter after long team Storage was tesed as follows: A PLGAMactige (90:10 PLGAtctide monomer) delivery system contining live bone cells having “sandwich” structure was prepared according tothe medhods of Example 1. The call-conaing system Was placed ina sterile pase vil and cooled down trate of 1" C/min to ~T0" C. using a feezing container (Nalgeae Cryo 1” C. fieeing conaines). The plane vist coniining the frozen cell delivery sysem was maintained at =10" C. for 4 hours. Te vial was then removed from the coniainer and inserted into liquid ritrogen for long term preservation. The vil was kept in long term preservation for least 48 hous. ‘The vial was then emoved from the lguid nitrogen and the cell deivery system was plced ina pet dish containing Sls of minimum essen medium at room temperature. ‘The pet sh was then placed in an incubator, which was imaiatsined at 37° C. ina flly-humidied atmosphere having 38 CO, in si. Optical microscopy was used monitor the cell release ativity. An optical microscopic photograph, taken 3 days after the stat of incubation, is Shown in FIG. 12. Aroview of FIG. 12 shows that significant postion of living ells was released from the delivery system in 3 day Example 5 ‘The wetability of a cell solution on the surface of an cleetrospun membrane was evaluated as follows: A PLGA! lactide (90:10 PLGA: lactde monomer) membrane was prepare in the manner described in Example 1. Approxi- ‘mately $ pls ofa cel solution containing live bone cells was placed onto the surface of the membrane using an eye Gropper and the contact angle of the droplet was measured fs a function of dime. ‘The test was repeated using a membrane that had first been dipped into a medium solution, The result of the two tests are shown graphically in FIG. 13, ‘A review of FIG. 13 reveals that, without pro-wetting the ‘membrane, the contact angle sats out at about 95°. After pre-wetting the membrane, the contact angle decreases to about 60°. Thus, pre-wetting the membrane significantly improves its wettability forthe cell solution, ‘Thus, while there has been disclosed what is presently believed to be preferred embodiments ofthe invention, those stulled in the art will appreciate that other and further changes and modifications ean be made without departing from the scope or spirit ofthe invention, and itis intended that all such other changes and modifications are included in and are within the scope of the invention as described in the appended claims. We claim: 1. A call delivery system comprising a biodegradable andior bioabsorbable fbrous matrix containing atleast about 20 weight percent of fibers having fiber diameters in the range of about 10 up to about 1000 nanometers of biodegradable and/or bioabsorbable fberizable material, and viable cells physically associated with said matrix as a carrer whereby said cells are contained and released at a controled rate 2. A cell delivery system acconding to claim 1, wherein said viable cells are issue precursor cells selected from the