All The Claims

I claim:

1. A container for use in making popcorn in a microwave oven, said container comprising:
a) An outer enclosure enclosing an outer cavity, said outer enclosure made from a rigid or semi-rigid microwave transparent material;
b) An inner enclosure enclosed within said outer cavity and enclosing an inner cavity, said inner enclosure including a base portion with a susceptor for absorbing microwave radiation to heat popcorn kernels located in the vicinity of said susceptor, said inner enclosure having an opening sealed by a seal;
c) a plurality of un-popped popcorn kernels and a charge enclosed in said inner cavity, wherein at least some of said plurality of kernels will pop and expand when said container is subjected to microwave radiation, and wherein the pressure in said inner cavity will increase and said seal will be released to release said popped kernels from said inner enclosure within said outer cavity;
said outer enclosure and said inner enclosure being adapted such that said outer enclosure is movable, by application of external forces to said outer enclosure, from a first configuration wherein said outer enclosure and said inner enclosure are in a generally flattened configuration to a second configuration wherein said outer enclosure is in an erected configuration and said outer cavity has increased in size so as to accommodate said popped kernels released from said inner cavity.
2. A container for use in making popcorn in a microwave oven, said container comprising:
a) a self erecting container made from a microwave transparent material and having an expandable internal cavity, said container being movable from a first generally flattened configuration to a second erected configuration, whereby the size of said cavity is increased;
b) an inner pouch containing a plurality of popcorn kernels and an associated charge, said pouch having a susceptor for absorbing microwave radiation to heat said kernels, and said pouch being located within said expandable cavity, wherein at least some of said plurality of kernels will pop and be released by said pouch within said cavity, when said container is subjected to microwave radiation.
3. A container as claimed in claim 1 wherein said microwave transmissible material is a cardboard.
4. A container as claimed in claim 1 wherein said cardboard is coated on at least one side with a grease resistant coating.
5. A container as claimed in claim 1 wherein said seal at said opening is formed with a heat activated adhesive, such that when said container is subjected to microwave radiation, said adhesive will soften to release said seal.
6. A container as claimed in claim 1 wherein said inner enclosure is formed with a generally rectangular or square configuration and is sealed on three sides thereof.
7. A container as claimed in claim 1 wherein said outer enclosure has a base and said inner enclosure is affixed to said base.
8. A container as claimed in claim 6 wherein said outer enclosure has a square or rectangular base and a side wall surrounding and extending up from said base, and wherein said inner enclosure is affixed to said base at a bottom panel, and said bottom panel extends beyond said base of said outer enclosure to provide a trough above said base.
9. A container as claimed in claim 1 wherein said outer enclosure has a base and a side wall surrounding and extending up from said base, and wherein said inner enclosure is affixed to said base at a bottom panel, and said bottom panel extends beyond said base of said outer enclosure to provide a trough above said base.
10. A method of forming a pouch for use in a microwave popcorn container from a longitudinally extending web, said web comprising:
a) A longitudinally extending base layer, said base layer having a longitudinally extending medial folding line;
b) A susceptor material bonded to and stretching along one half of a first side of said base layer;
c) A first strip of heat activated adhesive located on said opposite side to said first side, and extending longitudinally, proximate an upper edge on said opposite side of said base layer;
d) A series of transversely extending strips of heat activated adhesive located on said opposite side, said strips extending from a lower edge located opposite to said upper edge, to a position past said folding line;
said method comprising the steps of:
i. moving said web longitudinally
ii. folding said web about said medial folding line such that said upper portion of said opposite surface faces said lower portion of said opposite surface
iii. activating said heat adhesive to bond said lower portion to said upper portion along said transverse strips of adhesive to form a plurality of pockets closed along a bottom and both opposite side of said pockets
iv. moving said pockets horizontally with the open side of each of said plurality of pockets generally oriented upwards
v. filling each of said pockets with a plurality of un-popped popcorn kernels and a charge
vi. sealing the upper side of said pocket
vii. transversely cutting each of said pockets through at a medial position along said side seals to cut a plurality of sealed pouches from said web.
11. A method of forming a pouch for use in a microwave popcorn container from a longitudinally extending web, said web comprising:
a) A longitudinally extending base layer, said base layer having a longitudinally extending medial folding axis and a first side and a second opposite side;
b) A susceptor material secured to at least a portion of said first side said base layer;
said method comprising the steps of:
i. moving said web substantially horizontally;
ii. folding said web transversely about said longitudinally extending medial axis such that an upper portion of said opposite surface faces a lower portion of said opposite surface
iii. bonding said lower portion at discrete locations to form a plurality of pockets, said pockets being closed along a bottom edge and along opposite side edges of said pockets, and having an upwardly directed opening;
iv. moving said pockets horizontally to a filling station and filling each of said pockets through said opening with un-popped popcorn kernels and a charge;
v. sealing the opening of said pocket
vi. transversely cutting said web at a side edge of each of said pockets to cut a plurality of sealed pouches from said web.
12. A web for use in forming a pouch to hold popcorn kernels, said web comprising:
a) A longitudinally extending base layer, said layer having a longitudinally extending medial line;
b) A susceptor material bonded to and stretching along one half of a first side of said base layer;
c) A first strip of heat activated adhesive located on said opposite side to said first side, and extending longitudinally, proximate an upper edge on said opposite side of said base layer;
d) A series of transversely extending strips of heat activated adhesive located on said opposite side, said strips extending from a lower edge located opposite to said upper edge, to a position past said medial line.
13. A web for use in forming a pouch to hold popcorn kernels, said web comprising:
a) A longitudinally extending base layer having a first and second side, said layer having a longitudinally extending medial line and being made from a foldable material;
b) A susceptor material stretching along one half of said first side of said base layer;
c) A continuous strip of heat activated adhesive located on said second side which is opposite to said first side, and extending from a first position on said medial line, toward an upper edge of said web and thereafter to a second position on said medial line.
14. A pouch comprising:
a) A longitudinally extending base layer having a first outer side and a second inner side, said layer having a longitudinally extending medial fold line and being made from a foldable material;
b) A susceptor material positioned on a portion of said first side of said base layer;
c) A continuous strip of heat activated adhesive located on said second inner side which is opposite to said first side, and extending from a first position on said medial line, toward an upper edge of said web and thereafter to a second position on said medial line, said strip of adhesive bonding a first portion of said inner side to a second portion of said inner side to provide a cavity having a boundary defined by said medial fold line and said strip of adhesive;
d) A plurality of kernels and a charge contained within said cavity;
wherein said adhesive strip will release popcorn formed within said cavity, when said pouch is subjected to microwave radiation.
15. A method of forming a container for use in creating popcorn in a microwave oven, said container comprising an outer enclosure made from a blank and enclosing therein a pouch, said container being adapted to be movable with said pouch enclosed therein from a first substantially flattened configuration to a second erected configuration, said method comprising:
a) moving a longitudinally extending web of blanks to a pouch placement station, each blank having a plurality of panels;
b) placing a releasable pouch containing a plurality of popcorn kernels and a charge, on one of said plurality of panels
c) folding and securing said panels to enclose each said pouch within a cavity formed within said blank.
16. A container for use in making popcorn in a microwave oven, said container comprising:
a) A rigid or semi-rigid, outer housing made from a microwave transmissible material, said housing defining and enclosing an internal cavity; said housing having a tab portion protruding therefrom which is suitable for holding said container;
b) a plurality of popcorn kernels and an associated charge in said cavity,
c) a susceptor for absorbing microwave radiation to heat said kernels, wherein at least some of said plurality of kernels will pop and be released by said pouch within said cavity, when said container is subjected to microwave radiation.
17. A container as claimed in claim 16 wherein said housing is constructed with at least two housing portions joined together with at least one overlapping housing section positioned proximate said tab portion.
18. A container as claimed in claim 17 wherein said tab portion is attached to a removable lid portion, said tab portion permitting said lid portion to be readily opened.
19. A container as claimed in claim 18 wherein said tab portion is receivable in a slot in said housing to retain said lid in a closed position.
20. A container for making popcorn in a microwave open, comprising:
a) an outer enclosure having a first, low volume configuration and a second high volume configuration;
b) a pouch within said outer enclosure, said pouch containing popcorn kernels.
21. The container of claim 20 further comprising a latch for latching said outer enclosure in said high volume configuration.
22. The container of claim 20 wherein said outer enclosure has a pair of side walls which, in said high volume configuration are upstanding, said pair of side walls having co-operating features which latch together in said high volume configuration in order to maintain said outer enclosure in said high volume configuration.
23. The container of claim 22 wherein said pouch is adhered to a base wall of said outer enclosure.
24. The container of claim 23 wherein said pouch extends beyond a periphery of said base wall such that, in said high volume configuration, edges of said pouch extend away from said base wall.
25. The container of claim 20 wherein said container further comprises a susceptor.
26. The container of claim 25 wherein said pouch also contains a charge to facilitate popping of said kernels.
27. The container of claim 20 wherein said pouch has a closed opening which opens when said kernels are popping under the influence of a source of microwave heating.
28. The container of claim 27 wherein said opening is closed by an adhesive of a type which releases in the presence of heat.
29. The container of claim 27 wherein said outer enclosure is formed of cardboard.
30. A container for use in making popcorn in a microwave oven, said container comprising:
a) An outer enclosure defining an outer cavity, said outer enclosure made from a rigid or semi-rigid microwave transparent material;
b) An inner enclosure enclosed within said outer cavity and defining an inner cavity, said inner enclosure having a sealed opening;
c) a susceptor for absorbing microwave radiation to heat popcorn kernels located in the inner enclosure;
d) a plurality of un-popped popcorn kernels and a charge enclosed in said inner cavity, wherein at least some of said plurality of kernels will pop and expand when said container is subjected to microwave radiation, and wherein the pressure in said inner cavity will increase a seal at said sealed opening will be released to release said popped kernels from said inner enclosure to said outer cavity;
said outer enclosure and said inner enclosure being adapted such that said outer enclosure is movable, by application of external forces to said outer enclosure, from a first configuration wherein said outer enclosure and said inner enclosure are in a generally flattened configuration to a second configuration wherein said outer enclosure is in an erected configuration and said outer cavity has increased in size so as to accommodate said popped kernels released from said inner cavity.
31. A container for use in making popcorn in a microwave oven, said container comprising:
a) a self erecting outer enclosure having an internal cavity, said outer enclosure being movable from a first configuration to a second erected configuration, whereby the volume of said internal cavity is increased in said second erected configuration;
b) an inner enclosure containing a plurality of un-popped popcorn kernels and said inner enclosure being located within said expandable internal cavity, said pouch inner enclosure being arranged for releasing popping kernels into said internal cavity, when said container is subjected to a source of heat.
32. A container as claimed in claim 31 wherein said inner enclosure also has a charge associated with said kernels.
33. A container as claimed in claim 32 further comprising a susceptor adapted to absorb heat energy from said heat source to heat said kernels.
34. A container as claimed in claim 33 wherein said source of heat is microwave radiation.
35. A container as claimed in claim 34 wherein said outer enclosure is made from a microwave transparent material.
36. A container as claimed in claim 35 wherein said susceptor is positioned in said internal cavity of said outer enclosure.
37. A container as claimed in claim 36 wherein said susceptor is formed as part of said inner enclosure.
38. A container as claimed in claim in claim 37 wherein said microwave transparent material is cardboard.
39. A container as claimed in claim 31 wherein said first configuration is a generally flattened configuration.
40. A container as claimed in claim 35 wherein said first configuration is a generally flattened configuration.
41. A container as claimed in claim 37 wherein said pouch is made at least in part from a layer of a polyester material.
42. A container as claimed in claim 41 wherein said polyester material is PET.
43. A container as claimed in claim 41 wherein said polyester has a metallic susceptor material deposited on a surface thereof.
44. A container as claimed in claim 37 wherein said susceptor is positioned in said internal cavity adjacent a base panel of said outer enclosure.
45. A container as claimed in claim 38 wherein said cardboard is coated on at least one side with a grease resistant coating.
46. A container as claimed in claim 35 wherein said inner enclosure is constructed to be generally flexible.
47. A container as claimed in claim 46 wherein said inner enclosure is made with an opening formed between two parts of said inner enclosure, said opening being sealed with a heat activated adhesive before said inner enclosure is subjected to heating by microwave radiation, such that when said container is subjected to microwave radiation, said heat activated adhesive will soften to release said seal and allow said opening to open.
48. A container as claimed in claim 45 wherein said inner enclosure is formed with a generally rectangular or square configuration and is sealed with heat activated adhesive on three sides thereof, such that when said container is subjected to microwave radiation, an opening is formed on three sides of said pouch to release said popcorn into said internal cavity.
49. A container as claimed in claim 44 wherein said outer enclosure has a base and said inner enclosure is affixed to said base.
50. A container as claimed in claim 32 wherein said outer enclosure has a square or rectangular base and side walls surrounding and extending up from said base, and wherein said inner enclosure is affixed to said base at a bottom panel, and wherein said bottom panel has a trough to inhibit seepage of any part of the charge out of said cavity.
51. A container as claimed in claim 50 wherein said bottom panel has upwardly directed portions mating with upwardly directed portions of a mating top panel, said upwardly directed portions forming tab portions around a periphery of said inner enclosure, said upwardly directed portions of said bottom panel at least partially defining said trough.
52. A container as claimed in claim 32 wherein said outer enclosure has a square or rectangular base with four corners, and a side wall surrounding and extending up from said base, and wherein said inner enclosure is affixed to said base at a bottom panel, and said base has a barrier at, at least some of said corners to provide a trough so as inhibit the passage of grease or the like from the internal cavity.
53. A container as claimed in claim 32 wherein said outer enclosure is adapted so that said container is self-supporting when said outer enclosure is in said erected configuration.
54. A container as claimed in claim 32 where said outer enclosure comprises:
a) a top panel, a rear wall panel, a base panel and a front wall panel, each of said top, rear, base and front panels, being interconnected at transverse fold lines, in series, along at least one transverse edge and each of said top, rear, base and front panels having a first side edge and a second opposite side edge, each of said first side edges of said top, rear base and front panel being in general longitudinal alignment with each other, and each of said second side edges of said top, rear base and front panel being in general longitudinal alignment with each other;
b) a plurality of side wall panels, each of said top, rear, base and front panels having a side wall panel interconnected at longitudinal fold lines along each of said first and second side edges;
said side panels of said top, rear, base and front panels, co-operating to provide an outer enclosure adapted for movement between said first and second configurations.
55. A container as claimed in claim 54 wherein said side panels connected to said top panel have a tab portion and a notch, and wherein said side panels connected to said bottom panel have a tab portion and a notch, on each side of said top and bottom panel, said tab portions of said related side panels of one of said top and bottom panels, interconnecting with said notch portions of said other of said top and bottom panels, to provide an interlocking of side panels to define side walls of said container.
56. A container as claimed in claim 54 wherein side panels interconnected along said first side edge have features which interlock when said container is moved to said second configuration relationship for assisting in maintaining said outer enclosure in said second configuration.
57. A container as claimed in claim 54 wherein said top panel has one or more score lines defining the outline of an at least partially removable lid portion of said container.
58. A container as claimed in claim 54 wherein said top panel has a tab extension for holding said container.
59. A container as claimed in claim 58 wherein said front panel has a slot for receiving said tab extension to thereby temporary close said lid portion.
60. A method of forming a container for use in making popcorn in a microwave oven, said container comprising an outer enclosure made from a blank and enclosing therein a cavity, said cavity having therein an inner enclosure, said container having a susceptor and said container being adapted to be movable with said susceptor and said inner enclosure enclosed therein from a first configuration to a second erected configuration thereby increasing the volume of said cavity, said method comprising:
a) moving a blank to a pouch placement station, said blank having a plurality of panels, including a base panel;
b) at said pouch placement station, placing a releasable pouch containing a plurality of un-popped popcorn kernels and a charge, on said base panel;
c) folding and securing said panels to enclose said pouch within said cavity formed within said blank, to form a container capable of being moved from said first configuration to said second configuration.
61. A method as claimed in claim 60 wherein said pouch has an associated charge for said popcorn kernels.
62. An apparatus for forming a container for use in making popcorn in a microwave oven, said container comprising an outer enclosure made from a blank and enclosing therein a cavity, said cavity having therein a pouch, said container having a susceptor and said container being adapted to be movable with said susceptor and said inner enclosure enclosed therein from a first configuration to a second erected configuration thereby increasing the volume of said cavity, said apparatus comprising:
a) A magazine holding a plurality of blanks, each of said blanks having a plurality of panels, including a base panel;
b) A conveyor system for moving a blank from said blank magazine to a pouch placement station, and from said pouch placement station to folding and securing stations,
c) A pouch feeder positioned at said pouch placement station for placing a pouch containing a plurality of un-popped popcorn kernels on one of said base panel;
d) A folding and securing station operable to fold said panels to enclose said pouch within said cavity formed within said blank, so as to form a container capable of being moved from said first configuration to said second configuration.
63. A container for use in heating food products comprising:
a) an outer enclosure enclosing a cavity, said outer enclosure made from a rigid or semi-rigid microwave transparent material, said enclosure being formed from a plurality of interconnected panels, said plurality of panels including a plurality of medial panels including a base panel, and a plurality of upstanding side wall panels;
b) a gap between a first side wall panel and a second side wall panel of said plurality of side wall panels, said first and second side wall panels being positioned adjacent to each other and each interconnected to a medial panel,
c) a barrier positioned across said gap and extending between said first side wall panel and said second side wall panel and said base panel, said adapted to block said gap in the vicinity of said base panel to substantially prevent seepage through said gap of fluid held in said cavity.
64. A container as claimed in claim 63 further comprising:
a) an inner pouch containing a plurality of un-popped popcorn kernels and an associated charge and said pouch being located on said base panel and within said cavity, said pouch having an opening activated when said popcorn kernels are subjected to microwave radiation;
b) a susceptor for absorbing microwave radiation to heat said kernels,
wherein at least some of said plurality of kernels will pop and be released by said pouch within said expandable cavity, when said container is subjected to microwave radiation, and wherein said barrier inhibits the seepage of a part of said charge through said gap.
65. A container as claimed in claim 63 wherein said container is configured in the shape of a generally rectangular or square box having a top panel disposed opposite said base panel, and having first, second, third and fourth upstanding side wall panels disposed between said top panel and said base panel, said container having four corners at the abutment of the base plate and respectively, first and second, second and third, third and fourth and fourth and first side walls, with gaps being provided at the said four corners, said barrier located at said front corners to block said four gaps proximate said base, said barrier in abutment with said base, each said barrier being adapted to block said gap in the vicinity of said base panel to substantially prevent seepage through each said gap of fluid held in said cavity.

The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.

1. A hydraulic pump for a vehicle, comprising:
a block;
a pulley receiving an engine torque and rotating;
a pump housing attached to the block;
an outer rotor mounted in the pump housing;
an inner rotor mounted inside the outer rotor, and comprising gear teeth at an interior surface thereof;
a pinion gear engaged to the interior surface of the inner rotor;
a planet carrier connected to and rotating the pinion gear and comprising a carrier drive shaft, the carrier drive shaft comprising a first end fixedly connected to the pulley; and
a sun gear engaged to the pinion gear and comprising a sun gear drive shaft that is at least partially housed in the carrier drive shaft and is axially slidable;
wherein a first end of the sun gear drive shaft is attached to the carrier drive shaft under a first operating condition of an engine, and a second end of the sun gear drive shaft is attached to the block under a second operating condition of the engine.
2. The hydraulic pump of claim 1, further comprising an oil cylinder for axially sliding the sun gear drive shaft.
3. The hydraulic pump of claim 2, wherein the oil cylinder is mounted in the block.
4. The hydraulic pump of claim 1, further comprising a bearing at one side of the block, wherein the sun gear drive shaft penetrates and slides in the bearing.
5. The hydraulic pump of claim 4, further comprising an oil cylinder for axially sliding the sun gear drive shaft, wherein the oil cylinder operates when a rotational speed of an engine is larger than or equal to a predetermined value.
6. The hydraulic pump of claim 5, wherein the first end of the sun gear drive shaft is fixed to and rotates together with the carrier drive shaft when the oil cylinder operates.
7. The hydraulic pump of claim 6, wherein the second end of the sun gear drive shaft is inserted and rotates in the bearing when the oil cylinder operates.
8. The hydraulic pump of claim 6, wherein the carrier drive shaft comprises a fixing portion at the first end of the carrier drive shaft, and an elastic member interposed between the first end of the carrier drive shaft and the first end of the sun gear drive shaft.
9. The hydraulic pump of claim 8, wherein the first end of the sun gear drive shaft is splined to the fixing portion of the carrier drive shaft when the oil cylinder operates.
10. The hydraulic pump of claim 8, wherein the sun gear drive shaft is disengaged from the fixing portion by an elastic force of the elastic member when the oil cylinder does not operate.
11. The hydraulic pump of claim 10, wherein the second end of the sun gear drive shaft penetrates the bearing and is fixed to the block when the oil cylinder does not operate.
12. The hydraulic pump of claim 11, wherein the second end of the sun gear drive shaft is splined to the block.

1. A system for removing a boiler tube stub comprising,
a means for removing material from an inner surface of the boiler tube stub for a removal length leaving a shell of a removal length with a new inner diameter larger than an original inner diameter of the boiler tube stub and a remaining length of the boiler tube stub, from which no material has been removed from the inner surface, of the original inner diameter, such that there is a shoulder at the intersection of the shell and remaining length of the boiler tube stub,
a plug with a plug body of a length greater than the removal length of the shell, said plug body having a first side, a second side and a diameter larger than the original inner diameter of the tube stub and smaller than the new inner diameter of the shell, and
a plug extension extending from the first side of the plug body having a diameter smaller than the original diameter of the boiler tube stub.
2. A method for removing a boiler tube stub comprising,
removing material from an inner surface of the boiler tube stub for a removal length, leaving a shell of a removal length with a new inner diameter larger than an original inner diameter of the boiler tube stub and a remaining length of the boiler tube stub, from which no material has been removed from the inner surface, of the original inner diameter, such that there is a shoulder at the intersection of the shell and the remaining length of the boiler tube stub,
selecting a plug with
a plug body of a length greater than the removal length of the shell, said plug body having a first side, a second side and a diameter larger than the original inner diameter of the tube stub and smaller than the new inner diameter of the shell, and
a plug extension extending from the first side of the plug body having a diameter smaller than the original diameter of the boiler tube stub.
inserting the plug into the shell such that the plug extension enters the remaining length of the boiler tube stub and the first side of the plug rests on the shoulder at the intersection of the shell and the remaining length of the boiler tube stub, and
exerting sufficient force on the second side of the plug body to punch out the boiler tube stub.

The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.

1. A biomatrix composition comprising collagen I, collagen IV, laminin, entactin, and tenascin.
2. The biomatrix composition of claim 1 further comprising fibronectin and proteoglycans.
3. The composition of claim 1 where collagen I is present in a concentration greater than a concentration of collagen IV, laminin, entactin and tenascin.
4. The composition of claim 1 where the two most abundant components are collagen I and collagen IV are present in a concentration greater than a concentration of collagen IV, laminin, entactin, and tenascin.
5. The composition of claim 2 where the collagen I is present in a range of 3546% by weight, collagen IV is present in a range of 19-33% by weight, laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
6. The composition of claim 2 where the collagen I is present at a concentration of 36% by weight, collagen IV is present at a concentration 24% by weight, laminin is present at a concentration 20% by weight, entacin is present at a concentration of 12.5% by weight, tenascin is present at a concentration 5% by weight, fibronectin is present at a concentration 1% by weight and proteoglycans are present at a concentration 1.0% by weight.
7. The composition of claim 2 where said proteoglycan is a heparin sulfate proteoglycan.
8. The composition of claim 1 where said composition is a native composition derived from a human starting material.
9. The composition of claim 1 where said composition is a native composition derived from a human basement membrane tissue.
10. The composition of claim 1 where said composition is a native composition derived from a human placental amnion.
11. The composition of claim 1 where the composition is essentially free of endogenous growth stimulants and proteolytic enzymes.
12. The composition of claim 11 where the growth factors are selected from the group consisting of EGF, NGF, FGF, bFGF, VEGF, HGF, BDNF, and GDNF.
13. The composition of claim 11 where the proteolytic enzyme is collagenase.
14. The composition of claim 1 further comprising a growth stimulant or a growth inhibitor.
15. The composition of claim 14 where the growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
16. The composition of claim 15 where the growth factor is selected from the group consisting of: EGF, PDGF, bFGF, FGF, VEGF, NGF, KGF, HGF, BDNF, neurotropin-3, neurotropin-4, CNF, GGF, GMF, GDNF, and a combination of any of the foregoing.
17. The composition of claim 1 further comprising an addition of at least one protein found in an extracellular matrix.
18. The composition of claim 17 where said protein is selected from the group consisting of: fibrin, vitronectin, collagen II, collagen III, collagen V, collagen VI, collagen VII, collagen VIII, osteopontin and a combination of any of the foregoing.
19. The composition of claim 1 where said composition is capable of supporting growth of a non-transformed target cell type without inducing differentiation.
20. The composition of claim 19 where the target cell type is selected from the group consisting of: epithelial cells, stem cells, endothelial cells, transgenic cells, liver cells and neural cells.
21. The composition of claim 20 where the stem cells are totipotent or pluripotent.
22. The composition of claim 1 where said composition is capable of supporting the controlled growth of transformed cell types.
23. The composition of claim 1 where said composition can be used for the study of physiological and pathological processes.
24. The composition of claim 23 where the physiological processes are selected from the group consisting of: tissue growth, tissue development, bone remodeling, wound healing, angiogenesis, reproduction, and aging.
25. The composition of claim 23 where the pathological processes are selected from the group consisting of: tumorigenesis, metastasis, angiogenesis, vascular dysfunction, arthritis and atherosclerosis.
26. The composition of claim 1 where said composition forms a gel over a temperature range of 4 degrees C. to 37 degrees C.
27. A biomatrix composition comprising collagen I, collagen IV, and laminin.
28. The composition of claim 27 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight.
29. The composition of claim 27 further comprising entacin and tenascin.
30. The composition of claim 29 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, and tenascin is present in a range of 3.5-7.0% by weight.
31. The composition of claim 29 further comprising fibronectin and proteoglycans.
32. The composition of claim 31 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
33. A biomatrix composition for promoting growth and differentiation of a target cell, said composition being derived from a plurality of placental amnions by a process comprising the sequential steps of:
a. processing a plurality of placental amnions by cleaning said amnions to remove an undesirable material, rinsing said amnions at least 1 time in PBS, rinsing said amnions in a first buffer comprising an ammonium hydroxide solution, and rinsing said amnions at least one time in PBS and drying said amnions.
b. solubilizing and homogenizing said amnions by dissecting said amnions into a plurality of pieces in an ice cold second buffer comprising 0.5M acetic acid, homogenizing said amnions in said second buffer comprising 0.5M acetic acid to produce a first homogenate uniform in appearance, raising the pH of the first homogenate to a pH of 2.0, and digesting the first homogenate with a proteolytic enzyme and incubating the proteolytic enzyme with the first homogenate at 4 degrees C. for at least 12 hours;
c. centrifuging said first homogenate to isolate a first supernatant and adjusting said first supernatant to a pH of 7.8;
d. extracting the first supematant by adjusting said first homogenate to a Tris-base concentration of 50 mM and a salt concentration of 4 M and incubating the first supernatant at 4 degree C. for at least 12 hours;
e. centrifuging said first supernatant to isolate a first pellet and resuspending said first pellet in a third buffer comprising 0.5M acetic acid to produce a first suspension and adjusting said first suspension to a pH of 7.8;
f. extracting the first suspension by adjusting said first suspension to a Tris-base concentration of 50 mM and a salt concentration of 4 M and incubating the first suspension at 4 degree C. for at least 3 hours;
g. centrifuging said first suspension to isolate a second pellet and resuspending said second pellet in a dialysis buffer comprising 0.5M acetic acid to produce a second and incubating said second suspension at 4 degree C. for at least 12 hours;
h. placing the second suspension in a dialysis bag and dialyzing said second suspension against a suitable dialysis buffer with multiple changes of the dialysis buffer at 4 degree C. for at least 24 hours to produce a dialysate; and
i. recovering the dialysate from step (h) and concentrating the dialysate by ultrafiltration, said final dialysate comprising collagen, collagen IV, laminin, entacin, and tenascin.
34. The composition of claim 33 further comprising fibronectin and proteoglycans.
35. The composition of claim 34 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
36. The composition of claim 33 where said amnions are frozen at \u221220 degrees C. prior to said homogenization step.
37. The composition of claim 33 where the proteolytic enzyme is pepsin and said incubating the pepsin with said first homogenate occurs for at least 24 hours.
38. The composition of claim 33 where step (d) is repeated at least one time.
39. The composition of claim 33 where the dialysis buffer comprises 5 mM acetic acid, 5 mM KCl and 135 mM NaCl.
40. The composition of claim 33 where step (h) further comprises a sterilizing amount of a sterilization reagent.
41. The composition of claim 40 where the sterilization reagent is chloroform.
42. The composition of claim 33 where the first buffer is PBS and said ammonium hydroxide is present at a concentration of at least 0.1N.
43. The composition of claim 33 where the second buffer and the third buffer are PBS.
44. A method for analyzing a physiological process of interest by culturing a population of target cells relevant to said physiological process cells in three dimensions, said method comprising:
a. layering on a substrate a sufficient concentration of a biologically active biomatrix composition to produce a biomatrix coated disk, said biomatrix comprising collagen L collagen IV, laminin, entacin, and tenascin;
b. incubating said biomatrix coated substrate with a quantity of target cells in a buffered cell-culture medium to produce a target cell culture;
c. culturing said target cell culture under defined conditions; and
d. observing at least one characteristic relevant to said physiological process of said target cell culture over time.
45. The method of claim 44 where the biomatrix further comprises fibronectin and proteoglycans.
46. The method of claim 45 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
47. The method of claim 44 where the physiological process is tissue growth, tissue development, bone remodeling, wound healing, angiogenesis, reproduction, or aging.
48. The method of claim 44 where the target cells are transformed cells or non-transformed cells.
49. The method of claim 44 where the target cells are epithelial cells, stem cells, endothelial cells, liver cells, transgenic cells or neural cells.
50. The method of claim 49 where the stem cells are totipotent or pluripotent
51. The method of claim 44 where the substrate is a microbead or a disc.
52. The method of claim 51 where said disk has a porosity and said biomatrix composition is present at a concentration of 3 to 5 mgml.
53. The method of claim 51 where said biomatrix composition is present at a concentration of 25 to 125 \u03bcgbead and said microbeads are incubated under gravity free conditions.
54. The method of claim 44 where the buffered cell culture medium further comprises a growth stimulant.
55. The method of claim 54 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
56. The method of claim 44 where the biomatrix composition further comprises a growth stimulant.
57. The method of claim 56 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
58. The method of claim 44 further comprising adding a compound capable of a positive regulation of said physiological process or a negative regulation of said physiological process and determining the effect of said compound on at least one characteristic of said physiological process exhibited by said endothelial cell population.
59. The method of claim 44 further comprising the addition of a transformed cell type for coculture with said target cell.
60. The method of claim 44 where said method is used to study a pathological process.
61. The method of claim 60 where said pathological process is selected from the group consisting of: tumorigenesis, metastasis, angiogenesis, vascular dysfunction, arthritis and atherosclerosis.
62. A method for analyzing angiogenesis by culturing a population of endothelial cells in three dimensions, said method comprising:
a. layering on a substrate a sufficient concentration of a biologically active biomatrix composition to produce a biomatrix coated microbead, said biomatrix comprising collagen I, collagen IV, laminin, entacin, and tenascin;
b. incubating said biomatrix coated substrate with a quantity of endothelial cells in a buffered cell-culture medium to produce a endothelial cell culture;
c. culturing said endothelial cell culture under defined conditions;
d. observing at least one angiogenic characteristic of said endothelial cell culture over time.
63. The method of claim 62 where the biomatrix further comprises fibronectin and proteoglycans.
64. The method of claim 63 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
65. The method of claim 62 where said at least one angiogenic characteristic of said endothelial cell population is selected from the group consisting of: cell activation, sprout formation, capillary formation, cell migration, cell morphology, cell differentiation and any combination of the foregoing.
66. The method of claim 62 where the substrate is a microbead or a disc.
67. The method of claim 66 where said disk has a porosity and said biomatrix composition is present at a concentration of 3 to 5 mgml.
68. The method of claim 66 where said biomatrix composition is present at a concentration of 25 to 125 \u03bcgbead microbeads and said microbeads are incubated under gravity free conditions.
69. The method of claim 62 where the endothelial cell population comprises a plurality of human umbilical vein endothelial cells.
70. The method of claim 62 where the buffered cell culture medium further comprises a growth stimulant.
71. The method of claim 70 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
72. The method of claim 62 where the biomatrix further comprises a growth stimulant
73. The method of claim 72 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
74. The method of claim 62 further comprising adding a compound capable of promoting or inhibiting angiogenesis and determining the effect of said compound on at least one angiogenic characteristic of said endothelial cell population.
75. The method of claim 74 where said at least one angiogenic characteristic of said endothelial cell population is selected from the group consisting of: cell activation, sprout formation, capillary formation, cell migration, cell morphology, cell differentiation and any combination of the foregoing.
76. A method of analyzing neurogenesis by culturing a population of cells of neural origin in three dimensions, said method comprising:
a. layering on a substrate a sufficient concentration of a biologically active biomatrix composition to produce a biomatrix coated disk, said biomatrix comprising collagen I, collagen IV, laminin, entacin, and tenascin;
b. incubating said biomatrix coated substrate with a quantity of cells of neural origin in a buffered cell-culture medium to produce a neural cell culture;
c. culturing said neural cell culture under defined conditions; and
d. observing at least one neurological characteristic of said neural cell culture over time.
77. The method of claim 76 where the biomatrix further comprises fibronectin and proteoglycans.
78. The method of claim 77 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
79. The method of claim 76 where said at least one neurological characteristic of said endothelial cell population is selected from the group consisting of: axon formation, neurite formation, cell activation, cell morphology, cell migration, cell differentiation and any combination of the foregoing.
80. The method of claim 76 where the substrate is a microbead or a disc.
81. The method of claim 80 where said disk has a porosity and said biomatrix composition is present at a concentration of 3 to 5 mgml.
82. The method of claim 80 where said biomatrix composition is present at a concentration of 25 to 125 \u03bcgbead and said microbeads are incubated under gravity free conditions.
83. The method of claim 76 where the cells of neural origin are selected from the group consisting of: stem cells, neurospheres, neurons, and a combination of any of the foregoing.
84. The method of claim 76 where the buffered cell culture medium further comprises a growth stimulant.
85. The method of claim 84 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
86. The method of claim 76 where the biomatrix further comprises a growth stimulant.
87. The method of claim 86 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
88. The method of claim 76 further comprising adding a compound capable of promoting or inhibiting neurogenesis and determining the effect of said compound on at least one neurological characteristic of said endothelial cell population.
89. The method of claim 88 where said at least one angiogenic characteristic of said endothelial cell population is selected from the group consisting of: axon formation, neurite formation, cell activation, cell morphology, cell migration, cell differentiation and any combination of the foregoing.
90. A method for treating spinal cord injury in an subject in need of such treatment, said method comprising:
a. Preparing a three-dimensional biomatrix scaffold, said biomatrix scaffold containing neural precursor and said biomatrix scaffold comprising collagen I, collagen IV, laminin, entacin, and tenascin; and
b. Introducing said biomatrix scaffold into a subject in need of such treatment.
91. The method of claim 90 where the biomatrix further comprises fibronectin and proteoglycans.
92. The method of claim 91 where the collagen I is present in a range of 35-46% by weight, collagen IV is present in a range of 19-33% by weight, and laminin is present in a range of 16-21% by weight, entacin is present in a range of 7.5 to 14.5% by weight, tenascin is present in a range of 3.5-7.0% by weight, fibronectin is present in a range of 0-2.0% by weight and proteoglycans are present in a range of 0 to 1.3%.
93. The method of claim 90 where the subject is a mammal.
94. The method of claim 90 where the subject is a human.
95. The method of claim 90 where the neural progenitor cells are neural stem cells.
96. The method of claim 95 where said stem cells are totipotent or pluripotent.
97. The method of claim 90 where said neural progenitor cells are neurospheres.
98. The method of claim 90 where said biomatrix scaffold is present at a concentration of 15 to 300 ug.
99. The method of claim 90 where said introducing is accomplished by an injection at the site of said spinal cord injury.
100. The method of claim 90 where the biomatrix scaffold further comprises a growth stimulant.
101. The method of claim 100 growth stimulant is a growth factor, a cytokine, a peptide factor or an organic compound.
102. The method of claim 90 where the biomatrix scaffold further comprises a plurality of an accessory cell type.
103. The method of claim 102 where the accessory cell type is a Schwann cell or a mesenchymal cell.