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Kevin T. Campbell, Dustin J. Hadley, David L. Kukis, Eduardo A. Silva
Alginate hydrogels allow for bioactive and sustained release of VEGF-C and VEGF-D for lymphangiogenic therapeutic applications
www.plosone.org
www.plosone.org
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(oxidized)Tj
3.6113 0 Td
(with)Tj
1.9899 0 Td
(sodium)Tj
3.2201 0 Td
(periodate)Tj
-33.8227 -1.2983 Td
(\(Sigma\).)Tj
3.5829 0 Td
(All)Tj
1.4003 0 Td
(oxidized)Tj
3.617 0 Td
(alginate)Tj
3.3392 0 Td
(solutions)Tj
3.8438 0 Td
(were)Tj
2.1203 0 Td
(dialyzed,)Tj
3.7417 0 Td
(sterile)Tj
2.6078 0 Td
(filtered,)Tj
3.2939 0 Td
(lyophilized)Tj
4.6147 0 Td
(and)Tj
1.7292 0 Td
(stored)Tj
2.7099 0 Td
(at)Tj
-36.6007 -1.3039 Td
(-20C.)Tj
2.8006 0 Td
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4.3426 0 Td
(were)Tj
2.126 0 Td
(prepared)Tj
3.8041 0 Td
(by)Tj
1.1848 0 Td
(reconstituting)Tj
5.8224 0 Td
(alginate)Tj
3.3391 0 Td
(in)Tj
1.0318 0 Td
(phosphate)Tj
4.337 0 Td
(buffered)Tj
3.5773 0 Td
(saline)Tj
2.5002 0 Td
(sup-)Tj
-34.8659 -1.2983 Td
(plemented)Tj
4.4446 0 Td
(with)Tj
2.0013 0 Td
(calcium)Tj
3.3619 0 Td
(and)Tj
1.7291 0 Td
(magnesium)Tj
4.8869 0 Td
(ions)Tj
1.9049 0 Td
(\(PBS++;)Tj
3.5546 0 Td
(Life)Tj
1.7404 0 Td
(Technologies\).)Tj
6.0945 0 Td
(Hydrogels)Tj
4.3426 0 Td
(were)Tj
-34.0608 -1.2983 Td
(prepared)Tj
3.804 0 Td
(to)Tj
1.0262 0 Td
(create)Tj
2.5965 0 Td
(a)Tj
0.6519 0 Td
(final)Tj
2.0013 0 Td
(concentration)Tj
5.8223 0 Td
(of)Tj
1.0148 0 Td
(2%)Tj
1.4513 0 Td
(\(w/v\))Tj
2.3698 0 Td
(polymer)Tj
3.5603 0 Td
(with)Tj
1.9956 0 Td
(75/25)Tj
2.4547 0 Td
(\(LMW/HMW\))Tj
6.2476 0 Td
(algi-)Tj
-34.9963 -1.3039 Td
(nate)Tj
1.9218 0 Td
(solution)Tj
3.4866 0 Td
(and)Tj
1.7291 0 Td
(8.4)Tj
1.3947 0 Td
(mg/mL)Tj
3.1918 0 Td
(of)Tj
1.0148 0 Td
(calcium)Tj
3.3618 0 Td
(sulfate)Tj
2.8006 0 Td
(\(Sigma\).)Tj
3.5887 0 Td
(The)Tj
1.7801 0 Td
(mixture)Tj
3.3902 0 Td
(was)Tj
1.7008 0 Td
(then)Tj
2.0183 0 Td
(dispensed)Tj
4.1782 0 Td
(into)Tj
-35.5575 -1.2982 Td
(rubber)Tj
2.9253 0 Td
(molds)Tj
2.6815 0 Td
(with)Tj
2.0013 0 Td
(approximately)Tj
5.9754 0 Td
(8)Tj
0.6916 0 Td
(mm)Tj
1.8596 0 Td
(diameter)Tj
3.787 0 Td
(and)Tj
1.7291 0 Td
(1.5)Tj
1.3947 0 Td
(mm)Tj
1.8595 0 Td
(height)Tj
2.7326 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1782 0 Td
(for)Tj
1.3833 0 Td
(at)Tj
-34.9282 -1.2983 Td
(least)Tj
1.9842 0 Td
(25)Tj
1.1679 0 Td
(minutes)Tj
3.4695 0 Td
(at)Tj
0.9468 0 Td
(room)Tj
2.4264 0 Td
(temperature)Tj
5.1307 0 Td
(to)Tj
1.0262 0 Td
(ensure)Tj
2.8743 0 Td
(full)Tj
1.525 0 Td
(gelation.)Tj
3.6397 0 Td
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1.6214 0 Td
(hydrogels)Tj
4.1045 0 Td
(with)Tj
2.0013 0 Td
(FITC-Dex-)Tj
-31.9179 -1.2983 Td
(tran,)Tj
2.0976 0 Td
(40)Tj
/F14 1 Tf
1.1678 0 Td
[()]TJ
/F6 1 Tf
(L)Tj
1.27 0 Td
(of)Tj
1.0148 0 Td
(FITC-Dextran)Tj
5.9867 0 Td
(\(35)Tj
2.0296 0 Td
(kDa;)Tj
2.1089 0 Td
(Sigma\))Tj
3.0218 0 Td
(was)Tj
1.7007 0 Td
(incorporated)Tj
5.4198 0 Td
(into)Tj
1.8369 0 Td
(alginate)Tj
3.3392 0 Td
(prior)Tj
2.2507 0 Td
(to)Tj
1.0261 0 Td
(gelation)Tj
-34.2706 -1.3039 Td
(\(1)Tj
1.0318 0 Td
(mg/mL\).)Tj
3.7587 0 Td
(For)Tj
1.627 0 Td
(the)Tj
/F11 1 Tf
1.4684 0 Td
[()12()]TJ
1.0148 0 Td
[()-13()14()7()20()]TJ
/F6 1 Tf
2.0466 0 Td
(studies,)Tj
3.2145 0 Td
(lysozyme,)Tj
4.1215 0 Td
(VEGF-C)Tj
3.7417 0 Td
(and)Tj
1.7291 0 Td
(VEGF-D)Tj
3.8154 0 Td
(\(R&D)Tj
2.6249 0 Td
(systems\))Tj
3.6397 0 Td
(loaded)Tj
-33.8341 -1.2982 Td
(hydrogels)Tj
4.1045 0 Td
(had)Tj
1.7064 0 Td
(5)Tj
/F14 1 Tf
0.6974 0 Td
[()]TJ
/F6 1 Tf
(L)Tj
1.2642 0 Td
(of)Tj
1.0148 0 Td
(protein)Tj
3.1521 0 Td
(\(100)Tj
/F14 1 Tf
1.9899 0 Td
[()]TJ
/F6 1 Tf
(g/mL)Tj
2.88 0 Td
(in)Tj
1.0318 0 Td
(PBS++\))Tj
3.3392 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4683 0 Td
(alginate)Tj
3.3392 0 Td
(prior)Tj
2.2507 0 Td
(to)Tj
1.0262 0 Td
(gelation)Tj
-32.9497 -1.2983 Td
(\(500)Tj
1.9842 0 Td
(ng/mL\).)Tj
3.4866 0 Td
(For)Tj
1.6271 0 Td
(the)Tj
/F11 1 Tf
1.4683 0 Td
[()12()]TJ
1.0148 0 Td
[()-13()15()3()]TJ
/F6 1 Tf
1.8198 0 Td
(CAM)Tj
2.4605 0 Td
(study,)Tj
2.6192 0 Td
(VEGF-A)Tj
3.7587 0 Td
(\(R&D)Tj
2.6249 0 Td
(systems\),)Tj
3.8607 0 Td
(VEGF-C)Tj
3.7417 0 Td
(and)Tj
1.7348 0 Td
(VEGF-D)Tj
3.8098 0 Td
(had)Tj
-36.0111 -1.3039 Td
(20)Tj
/F14 1 Tf
1.1678 0 Td
[()]TJ
/F6 1 Tf
(L)Tj
1.2699 0 Td
(of)Tj
1.0148 0 Td
(growth)Tj
3.0728 0 Td
(factor)Tj
2.5398 0 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4683 0 Td
(alginate)Tj
3.3392 0 Td
(prior)Tj
2.2564 0 Td
(to)Tj
1.0205 0 Td
(gelation)Tj
3.4129 0 Td
(\(2)Tj
/F14 1 Tf
1.0374 0 Td
[()]TJ
/F6 1 Tf
(g/mL\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 351.4393 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Proliferation)Tj
5.485 0 Td
(assay)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 334.4881 cm
BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(LECs)Tj
2.3527 0 Td
(\(P7\))Tj
1.9332 0 Td
(were)Tj
2.1203 0 Td
(seeded)Tj
2.9083 0 Td
(at)Tj
0.9525 0 Td
(10,000)Tj
2.8233 0 Td
(cells/cm)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 363.6283 338.9102 cm
BT
/F6 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(2)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 369.411 334.4881 cm
BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(in)Tj
1.0374 0 Td
(12-well)Tj
3.1181 0 Td
(tissue)Tj
2.4605 0 Td
(culture)Tj
3.0387 0 Td
(plates)Tj
2.5001 0 Td
(with)Tj
1.9956 0 Td
(serum-free)Tj
-31.0901 -1.3039 Td
(EBM-2)Tj
3.0841 0 Td
(and)Tj
1.7348 0 Td
(allowed)Tj
3.2881 0 Td
(to)Tj
1.0262 0 Td
(adhere)Tj
2.9253 0 Td
(for)Tj
1.389 0 Td
(16)Tj
1.1679 0 Td
(hours.)Tj
2.7381 0 Td
(After)Tj
2.2904 0 Td
(serum)Tj
2.7212 0 Td
(deprivation,)Tj
5.0627 0 Td
(conditional)Tj
4.7962 0 Td
(medias)Tj
3.05 0 Td
(were)Tj
-35.274 -1.2982 Td
(added)Tj
2.6589 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4684 0 Td
(cells)Tj
1.9218 0 Td
(including)Tj
4.0422 0 Td
(EGM-2MV)Tj
4.8076 0 Td
(without)Tj
3.3334 0 Td
(growth)Tj
3.0727 0 Td
(factors)Tj
2.9027 0 Td
(supplemented)Tj
5.8563 0 Td
(with)Tj
1.9956 0 Td
(VEGF-C)Tj
-33.0857 -1.2983 Td
(or)Tj
1.0942 0 Td
(VEGF-D)Tj
3.8154 0 Td
(at)Tj
0.9524 0 Td
(various)Tj
3.1465 0 Td
(concentrations)Tj
6.1795 0 Td
(\(0,)Tj
1.2585 0 Td
(10,)Tj
1.3946 0 Td
(20,)Tj
1.4003 0 Td
(40,)Tj
1.3946 0 Td
(50,)Tj
1.4003 0 Td
(80)Tj
1.1679 0 Td
(or)Tj
1.0998 0 Td
(100)Tj
1.6441 0 Td
(ng/mL\).)Tj
3.4866 0 Td
(After)Tj
2.2847 0 Td
(4)Tj
0.6973 0 Td
(days)Tj
1.9956 0 Td
(with)Tj
-34.4123 -1.3039 Td
(daily)Tj
2.1487 0 Td
(media)Tj
2.6929 0 Td
(changes,)Tj
3.6283 0 Td
(the)Tj
1.4683 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(detached)Tj
3.8096 0 Td
(with)Tj
1.9956 0 Td
(0.05%)Tj
2.6306 0 Td
(Trypsin-EDTA)Tj
6.2985 0 Td
(\(Invitrogen\).)Tj
5.3518 0 Td
(The)Tj
-34.0665 -1.2983 Td
(total)Tj
2.0013 0 Td
(number)Tj
3.4185 0 Td
(of)Tj
1.0148 0 Td
(cells)Tj
1.9219 0 Td
(were)Tj
2.1203 0 Td
(quantified)Tj
4.3086 0 Td
(via)Tj
1.3777 0 Td
(a)Tj
0.6519 0 Td
(Countess)Tj
3.9117 0 Td
(automated)Tj
4.4901 0 Td
(cell)Tj
1.559 0 Td
(counter)Tj
3.3222 0 Td
(\(Life)Tj
2.0749 0 Td
(Technolo-)Tj
-32.1729 -1.2982 Td
(gies\))Tj
2.075 0 Td
(and)Tj
1.7291 0 Td
(averaged)Tj
3.753 0 Td
(per)Tj
1.5364 0 Td
(condition)Tj
4.1159 0 Td
(\(n)Tj
1.1055 0 Td
(=)Tj
0.7993 0 Td
(56\).)Tj
2.2563 0 Td
(The)Tj
1.7802 0 Td
(data)Tj
1.9162 0 Td
(was)Tj
1.6951 0 Td
(then)Tj
2.0182 0 Td
(normalized)Tj
4.7792 0 Td
(to)Tj
1.0261 0 Td
(the)Tj
1.4684 0 Td
(no)Tj
1.2756 0 Td
(growth)Tj
-33.3295 -1.304 Td
(factor)Tj
2.5398 0 Td
(control)Tj
3.1238 0 Td
(\(EGM-2MV)Tj
5.1477 0 Td
(without)Tj
3.3335 0 Td
(the)Tj
1.4683 0 Td
(addition)Tj
3.5999 0 Td
(of)Tj
1.0148 0 Td
(growth)Tj
3.0728 0 Td
(factorsN)Tj
4.1555 0 Td
(media\).)Tj
ET
Q
q
1 j
1 J
0 w
11.9999 0 0 11.9999 200.0125 217.0204 cm
BT
/F0 1 Tf
1 TL
-0.0033 Tc
0 0 Td
(Sprouting)Tj
4.4457 0 Td
(assay)Tj
ET
Q
q
1 j
1 J
0 w
10 0 0 10 200.0125 200.0125 cm
BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(Cytodex)Tj
3.5602 0 Td
(3)Tj
0.6917 0 Td
(microcarrier)Tj
5.2724 0 Td
(\(MC\))Tj
2.4548 0 Td
(beads)Tj
2.4775 0 Td
(\(GE)Tj
1.8425 0 Td
(Healthcare)Tj
4.5694 0 Td
(Life)Tj
1.7348 0 Td
(Sciences\))Tj
3.9061 0 Td
(were)Tj
2.126 0 Td
(prepared)Tj
3.804 0 Td
(and)Tj
1.7292 0 Td
(seeded)Tj
-34.1686 -1.3039 Td
(with)Tj
1.9955 0 Td
(LECs)Tj
2.3528 0 Td
(as)Tj
1.0148 0 Td
(previously)Tj
4.3483 0 Td
(described)Tj
4.0535 0 Td
(in)Tj
1.0375 0 Td
(detail)Tj
2.4151 0 Td
([)Tj
0.83 0.64 0.02 0 k
(26)Tj
0 g
(].)Tj
2.0749 0 Td
(Briefly,)Tj
3.0784 0 Td
(MC)Tj
1.7745 0 Td
(beads)Tj
2.4775 0 Td
(were)Tj
2.1203 0 Td
(hydrated)Tj
3.787 0 Td
(overnight)Tj
4.0819 0 Td
(at)Tj
-36.612 -1.2982 Td
(room)Tj
2.4207 0 Td
(temperature)Tj
5.1307 0 Td
(in)Tj
1.0375 0 Td
(DPBS)Tj
2.5681 0 Td
(while)Tj
2.3698 0 Td
(applying)Tj
3.6737 0 Td
(gentle)Tj
2.6248 0 Td
(agitation)Tj
3.7361 0 Td
(and)Tj
1.7291 0 Td
(were)Tj
2.126 0 Td
(then)Tj
2.0182 0 Td
(sterilized)Tj
3.8154 0 Td
(via)Tj
-33.2501 -1.2983 Td
(autoclaving.)Tj
5.0513 0 Td
(Following)Tj
4.2179 0 Td
(cooling,)Tj
3.4072 0 Td
(the)Tj
1.474 0 Td
(sterile)Tj
2.6079 0 Td
(MC)Tj
1.7801 0 Td
(beads)Tj
2.4718 0 Td
(were)Tj
2.1203 0 Td
(mixed)Tj
2.7213 0 Td
(with)Tj
2.0012 0 Td
(1.5)Tj
1.3946 0 Td
(mL)Tj
1.5761 0 Td
(of)Tj
1.0148 0 Td
(LECs)Tj
2.3527 0 Td
(\(~5)Tj
1.6158 0 Td
(x)Tj
-35.807 -1.2983 Td
(10)Tj
ET
Q
q
1 j
1 J
0 w
7.4999 0 0 6.6659 209.537 152.4472 cm
BT
/F6 1 Tf
1 TL
-0.0053 Tc
0 0 Td
(6)Tj
ET
Q
q
1 j
1 J
0 w
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BT
/F6 1 Tf
1 TL
-0.004 Tc
0 0 Td
(P7)Tj
1.2529 0 Td
(cells\))Tj
2.262 0 Td
(in)Tj
1.0375 0 Td
(EGM-2MV)Tj
4.8075 0 Td
(medium.)Tj
3.8324 0 Td
(The)Tj
1.7858 0 Td
(solution)Tj
3.4809 0 Td
(of)Tj
1.0148 0 Td
(beads)Tj
2.4718 0 Td
(and)Tj
1.7292 0 Td
(LECs)Tj
2.3527 0 Td
(were)Tj
2.126 0 Td
(transferred)Tj
4.6374 0 Td
(to)Tj
1.0262 0 Td
(a)Tj
-35.3421 -1.3039 Td
(FACS)Tj
2.5681 0 Td
(tube)Tj
1.9786 0 Td
(and)Tj
1.7291 0 Td
(incubated)Tj
4.1782 0 Td
(at)Tj
0.9525 0 Td
(37C)Tj
2.228 0 Td
(for)Tj
1.3833 0 Td
(4)Tj
0.6973 0 Td
(hours)Tj
2.5115 0 Td
(with)Tj
2.0012 0 Td
(gentle)Tj
2.6249 0 Td
(agitation)Tj
3.736 0 Td
(applied)Tj
3.1578 0 Td
(by)Tj
1.1792 0 Td
(inverting)Tj
3.8664 0 Td
(the)Tj
-34.7921 -1.2982 Td
(tube)Tj
1.9728 0 Td
(35)Tj
1.6895 0 Td
(times)Tj
2.3924 0 Td
(every)Tj
2.3471 0 Td
(20)Tj
1.1678 0 Td
(minutes.)Tj
3.6964 0 Td
(The)Tj
1.7801 0 Td
(LEC)Tj
1.9899 0 Td
(seeded)Tj
2.9084 0 Td
(MC)Tj
1.7744 0 Td
(beads)Tj
2.4718 0 Td
(were)Tj
2.126 0 Td
(then)Tj
2.0183 0 Td
(cultured)Tj
3.5659 0 Td
(in)Tj
1.0375 0 Td
(T25)Tj
1.7801 0 Td
(flasks)Tj
-34.7184 -1.2983 Td
(on)Tj
1.2756 0 Td
(a)Tj
0.6519 0 Td
(rotating)Tj
3.4129 0 Td
(shaker)Tj
2.8289 0 Td
(\(~1)Tj
1.6158 0 Td
(rev/sec\))Tj
3.3505 0 Td
(at)Tj
0.9524 0 Td
(37C)Tj
2.228 0 Td
(with)Tj
1.9956 0 Td
(daily)Tj
2.1487 0 Td
(media)Tj
2.6929 0 Td
(changes)Tj
3.4015 0 Td
(until)Tj
2.109 0 Td
(approaching)Tj
5.2327 0 Td
(100%)Tj
-33.8964 -1.3039 Td
(confluent.)Tj
4.2519 0 Td
(Confluent)Tj
4.2689 0 Td
(LEC)Tj
1.9899 0 Td
(seeded)Tj
2.9027 0 Td
(MC)Tj
1.7745 0 Td
(beads)Tj
2.4718 0 Td
(were)Tj
2.1203 0 Td
(then)Tj
2.0125 0 Td
(incorporated)Tj
5.4142 0 Td
(within)Tj
2.8119 0 Td
(fibrin)Tj
2.4662 0 Td
(gels)Tj
1.7121 0 Td
(as)Tj
1.0148 0 Td
(previ-)Tj
-35.2117 -1.2983 Td
(ously)Tj
2.313 0 Td
(described)Tj
4.0535 0 Td
([)Tj
0.83 0.64 0.02 0 k
(24)Tj
0 g
(,)Tj
0.83 0.64 0.02 0 k
1.7405 0 Td
(27)Tj
0 g
(].)Tj
1.7347 0 Td
(Briefly,)Tj
3.0728 0 Td
(MC)Tj
1.7801 0 Td
(beads)Tj
2.4718 0 Td
(were)Tj
2.1203 0 Td
(suspended)Tj
4.4391 0 Td
(in)Tj
1.0374 0 Td
(N)Tj
0.9525 0 Td
(media)Tj
2.6929 0 Td
(and)Tj
1.7348 0 Td
(combined)Tj
4.2406 0 Td
(with)Tj
ET
Q
q
1 j
1 J
0 w
576 737.1 m
36 737.1 l
36 737.6 l
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