ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH., 29(1), 57–70 (2001)

A SYSTEM ESTABLISHINGCOMPATIBILITY PROFILES FORARTIFICIAL OXYGEN CARRIERS

AND OTHER SUBSTANCES

Stephanie Dinkelmann,1 Wolfgang Rohlke,2

Hasso Meinert,2 and Hinnak Northoff1

1Institut fur Transfusionsmedizin, Otfried-Mueller-Str. 4/1,D-72076 Tubingen, Germany

2AG Chemie Biokompatibler Verbindungen,Universitat Ulm, D-89069 Ulm, Germany

ABSTRACT

Worldwide, great efforts are being made to develop a clini-cally useful artificial oxygen carrier. Toxicological and immunolog-ical compatibility is generally tested using animal experiments butinflammatory parameters in particular show large species-specificdifferences. Therefore, we developed an in vitro system using hu-man components to establish a compatibility profile of unknowncompounds. The test system comprises induction of hemolysis, ac-tivation of complement (C3a), induction/suppression of cytokineproduction, influence on cell proliferation, direct toxicity on pe-ripheral leukocytes, and phagocytosis of the material under testand of microbes. The test system will be described, along with re-sults of various perfluorocarbon emulsions. When testing lecithin-based perfluorodecalin (PFD) emulsions, and comparing them toPluronic-based PFD emulsions, we could show that Pluronic-basedemulsions were virtually untoxic to peripheral human leukocytes.

57

Copyright C© 2001 by Marcel Dekker, Inc. www.dekker.com

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58 DINKELMANN ET AL.

They neither inhibited cell proliferation nor caused any hemolysis,but caused mild to moderate inhibition of endotoxin-induced cy-tokine production. At the same time, lecithin-based PFD emulsioncaused substantial cytotoxicity in phagocytic cells like monocytes(60–100% after 24 h incubation) and granulocytes (10–20% after24 h incubation). They also suppressed endotoxin-induced cytokineproduction in monocytes to more than 98% and inhibited cell pro-liferation of an endothelial (ECV 304) and a monocytic cell line(MonoMac6) to more than 95%.

INTRODUCTION

During and after the development of artificial oxygen carriers designed forpotential use in humans, and following analysis of their physicochemical proper-ties, we see the importance of determining their biocompatibility profile in vitrobefore they may be tested in animals or humans. On the one hand, many extensiveanimal experiments could be saved; on the other hand, using tests based on humancells may provide a better predictive value for the judgement of in vivo compati-bility in humans. It is possible that a substance can be shown to be intolerable inthe animal model but be without major problems for use in humans. Much morelikely, and abundantly evident from the literature, are situations where extensiveexperiments with rodents seemed to indicate compatibility and beneficial effects,whereas ensuing human trials ended in severe disappointment. Therefore we de-veloped a multitask test system exploring the interference of test substances withvital functions on the cellular level (Fig. 1). We gathered information on directtoxic effects as well as influences on several cellular functions.

To estimate direct cytotoxicity, data will show the degree of hemolysis in-duced by different perfluorocarbon emulsions (PFC emulsions) and their effects onthe survival of peripheral human leukocytes. To determine the rate of dead leuko-cytes, we used flow cytometry. We prefer this method because many more cells canbe counted in relation to light microscopy. Furthermore, a discrimination betweenmonocytes, lymphocytes, and granulocytes is possible, and for each populationthe rate of dead cells can be measured separately. In addition to cytotoxicity, theinfluence of perfluorocarbon emulsions on cell proliferation is determined. We useonly human cell lines, and especially cells that will be first in contact with drugsfor intravenous application. Therefore, data will be presented with an endothelialcell line (ECV 304) and a monocytic cell line (MonoMac6). The cytokine re-lease is an important cellular function. We focused our tests on interleukin-1β andinterleukin-6, because these are pleiotropic factors that are released by activatedmonocytes, neutrophils, and several lymphocytes. They play a key role in defenseagainst infectious diseases, tissue damage, and inflammation. Massive induction

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ARTIFICIAL OXYGEN CARRIERS 59

Figure 1. A multitask system for biocompatibility testing of artificial oxygen carriers.

of these cytokines, or massive suppression of stimulan-induced cytokines, wouldbe reason for caution. The biocompatibility profile is completed by testing com-plement activation and phagocytosis. These data will be shown elsewhere.

We investigated various perfluorodecalin emulsions. Perfluorodecalin (PFD)possesses a high solubility for oxygen and carbon dioxide. PFD was developed tobe used as red cell substitute (1,3). PFD alone is not soluble in water and must beemulsified for intravenous application. The block polymer Pluronic and lecithin arewidely used as emulsifiers. The PFD/lecithin emulsion, the PFD/Pluronic emulsionand, in addition, perfluorodecalin and the emulsifiers alone, were tested. Lecithin isa common component of several pharmaceutical preparations (e.g., lipid solutionsfor intravenous application).

MATERIALS AND METHODS

Peripheral leukocytes, serum, and erythrocytes were obtained from healthyvolunteer donors.

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60 DINKELMANN ET AL.

Cell Lines

ECV 304 cells are human umbilical cord venous endothelial cells (4). ECV304 cells were purchased from the German Collection of Microorganisms and CellCultures (Braunschweig, Germany). The cells were cultured with Medium 199(Gibco, Eggenstein, Germany) supplemented with 20% heat-inactivated fetal calfserum (Roth, Karlsruhe). MonoMac6 are human acute monocytic leukemia cells(5). They were also purchased from the German collection. The culture mediumfor MonoMac6 contained: RMPI 1640 (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Myoclone Super Plus, Gibco) and 10 mL HEPESbuffer (Gibco) 2.5 mL nonessential amino acids (Gibco), 3.5 mL L-glutamin(Gibco), and 4.5 mL OPI (Sigma, Steinheim, Germany).

Test Substances

Ringer’s lactate (Baxter, Unterschleissheim, Germany) perfluorodecalin(PFD; Fluka # 77264); PFD emulsion with lecithin, containing 18.5% (w/v) per-fluorodecalin and 1.5% (w/v) perfluorodimorpholinopropane (FDMP, Eup0002),emulsified with 2.5% (w/v) lecithin (Lipoid E100�, Lipoid KG) in Ringer’s lactate,homogenized and steam sterilized; initial average droplet size: 130–239 nm; PFDemulsion with Pluronic, containing 18.5% (w/v) perfluorodecalin and 1.5% (w/v)perfluorodimorpholinopropane (FDMP, Eup 0002), emulsified with 4% (w/v)Pluronic PE 6800� (BASF, Ludwigshafen, Germany) in Ringer’s lactate, homog-enized and steam sterilized; initial average droplet size: 290–346 nm; lecithin-emulsion, containing 2.5% (w/v) lecithin (Lipoid E100, Lipoid KG) in Ringer’slactate; initial average droplet size: 222 nm; 4% (w/v) Pluronic PE 6800� (BASF,Ludwigshafen, Germany) in Ringer’s lactate.

Sterility Control

For all test substances, controls of sterility were employed each time beforeuse. The substances were plated onto blood/agar plates and incubated at 37◦C,5 vol.-% CO2 in humid atmosphere. After 24 and 48 h, the plates were controlled forbacterial growth. None of the substances used in the tests showed bacterial growth.

Hemolysis Assay

A 20% (w/v) erythrocyte suspension was prepared with Ringer’s lactate. Todetermine the hemolytic activity of the test substances, 50 µL of the erythrocyte

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ARTIFICIAL OXYGEN CARRIERS 61

suspension was incubated together with 100 µL of the test substance plus 100 µLof serum or isotonic saline. Erythrocyte suspension, together with isotonic saline,was used as negative control; erythrocyte suspension with distilled water was usedas positive control. The samples were incubated for 1 h at 37◦C in a water bath, thenthe samples were centrifuged for 2 min at 2.000g (Heraeus Instruments, Stuttgart,Germany) and the optical density of the supernatants were measured spectrophoto-metrically (SLT Labinstruments, Crailsheim, Germany). A test substance inducedhemolysis when the following quotient was greater than 2:

ODt+R.L./s+e

ODt+R.L./s + ODR.L./s+e(1)

where ODt+R.L./s+e is the optical density of the supernatant consisting of testsubstance (t) plus Ringer’s lactate or serum plus erythrocytes (e); ODt+R.L./s is theoptical density of the supernatant consisting of test substance plus Ringer’s lactateor serum; and ODR.L./s+e is the optical density of the supernatant consisting ofRinger’s lactate or serum plus erythrocytes.

Cytokine Assay

Mononuclear leukocytes were prepared with gradient separation (Lympho-flot separation medium, Biotest, Dreieich, Germany) using human blood (buffycoat from routine blood donations). The mononuclear leukocytes were used witha final concentration of 1 × 106 cells/mL and maintained with 5 vol.-% fetalcalf serum (Roth, Germany) and RPMI (1640, Gibco, Eggenstein, Germany).One hundred micro liters of the cell suspension were incubated for 4 h at 37◦C,5 vol.-% CO2, humid atmosphere, together with 50 µL of the test substance or thecontrols. Then lipopolysaccharide (Salmonella typhimurium, Difco, Augsburg,Germany) was added to a final concentration of 10 ng/mL and the cell suspensionwas incubated for another 20 h. Thereafter, the interleukin-1β and interleukin-6concentrations of the supernatant were measured using the ELISA technique(IL1β Quantikine, DPC, Bad Nauheim, Germany; IL6, Pharmingen, Hamburg,Germany). The degree of suppression or induction of interleukin-1β (IL1β) orinterleukin-6 (IL6) production was determined by the following quotients:

Degree of suppression = 1 − [IL1β(or IL6)]MNL+test compounds+LPS

[IL1β(or IL6)]MNL+LPS(2)

where [IL1β (or IL6)]MNL+test compounds+LPS equals concentration of IL1β (IL6)after incubation of mononuclear leukocytes (MNL) with various test compoundsplus lipopolysaccharide (LPS); and [IL1β(or IL6)]MNL+LPS equals concentration

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62 DINKELMANN ET AL.

of IL1β (IL6) after incubation of mononuclear leukocytes (MNL) with LPS.

Degree of induction = [IL1β(or IL6)]MNL+test compounds

[IL1β(or IL6)]MNL+LPS(3)

Cell Proliferation Assay

For this assay, 96-well culture plates (Falcon, Becton, Dickinson, Heidel-berg, Germany) were used. Each well contained 5 × 104 cells (ECV 304 or Mono-Mac6) in a volume of 100 µL. To each well were added 50 µL of a test substanceor the control (culture medium). The test substances and the controls were placedsymmetrically and alternately to avoid position effects on the microtiter plate.The culture plates were incubated for 24 h at 37◦C, 5 vol.-% CO2, humid atmo-sphere. Then 20 µL of 3H-thymidine (37 MBq/mL, Amersham) was added toeach well. After another 24 h of incubation at 37◦C, the culture plates were har-vested using an automated harvester. As the ECV 304 are adherent cells, cultureswere washed and trypsinized (trypsin from Biochrom, Berlin, Germany) beforeharvesting. Cell proliferation was determined by scintillation counting (Betaplate,LKB Wallac/Pharmacia, Finland). The relative 3H-thymidine incorporation wascalculated as follows:

rel.3H-thymidine incorporation tc [%]

=3H-thymidine incorporation tc [cpm]

3H-thymidine incorporation cm [cpm](4)

where tc is the test compound and cm is the culture medium.

Toxicity Assay

For peripheral leukocyte separation, anticoagulated (sodium-Heparin,10 I.E./mL) blood was incubated at ambient temperature for 1 h on a densitygradient (Ficoll 1077, Biochrom, Berlin, Germany) without centrifugation. There-after, the supernatant was obtained. For each sample, 75 µL leukocyte suspensionwas incubated together with 50 µL test substance at 37◦C, 5 vol.-% CO2, humidatmosphere. Samples were obtained after 0, 2, 4, 6, 8, 13, 17, and 24 h. The cellswere stained with CD45 FITC (panleukocyte marker, green fluorescence, BectonDickinson, Heidelberg, Germany), CD14 PE (monocyte marker, orange fluores-cence, Becton Dickinson), and propidium iodide (Sigma, Steinheim, Germany,DNA staining, determination of dead cells). The flow cytometric analyses wereperformed on a flow cytometer from Coulter (Coulter Epics XL, Coulter, Krefeld,Germany). Gates were set for the various leukocyte populations; 10,000 cells wereacquired; data were analysed using XL1 and XL2 software (Coulter).

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ARTIFICIAL OXYGEN CARRIERS 63

Table 1. Hemolysis Assay

Hemolysis in the Presence Hemolysis in theTest Compound of Isotonic Saline Solution Presence of Serum

Ringer’s lactate (n = 7) Negative NegativePerfluorodecalin (n = 7) Negative NegativeEmulsion lecithin (n = 4) Negative/weak positive NegativeEmulsion Pluronic (n = 5) Negative NegativeEmulsion PFD/lecithin (n = 4) Negative/weak positive NegativeEmulsion PFD/pluronic (n = 4) Negative Negative

PFD = Perfluorodecalin, n = number of measurements.

RESULTS

Hemolysis Assay

The results of the hemolysis tests are shown in Table 1. Only lecithin andthe lecithin/perfluorodecaline emulsion cause a weak positive hemolysis in thepresence of isotonic saline. In the presence of serum, none of the test compoundscaused a hemolysis.

Induction and Suppression of the Interleukin-1βProduction of Mononuclear Leukocytes

Lipopolysaccharide (LPS) is a strong stimulus for mononuclear leukocytes(MNL). After incubation of MNL with LPS, they produce (among others)interleukin-1β and interleukin-6. The results of our tests are depicted in Figure 2.We used Ringer’s lactate as control, because it is also a component of the PFCemulsions. The incubation of MNL with Ringer’s lactate resulted in a mild decreaseof interleukin-1β concentration. The incubation with perfluorodecalin (PFD) in-duced an average suppression of 26%, a decrease that is not significant. In contrast,lecithin and the PFD/lecithin emulsion suppressed the interleukin-1β concentra-tion to 98%. Finally, Pluronic caused a suppression of 46%. None of the testcompounds induced interleukin-1β by itself.

Induction and Suppression of the Interleukin-6Production of Mononuclear Leukocytes

The determination of the interleukin-6 concentrations after incubation ofmononuclear leukocytes with PFC emulsions and LPS showed similar results

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64 DINKELMANN ET AL.

Figure 2. Interleukin-1β release after incubation of mononuclear leucocytes with varioustest substances plus LPS. n = number of measurements; n = 7 for “none,” R.L., and PFD;n = 2 for lecithin emulsion, PFD/lecithin, and Pluronic emulsion.

(Fig. 3) in comparison to the interleukin-1β tests. With PFD, the interleukin-6production of mononuclear leukocytes is reduced by 22%. The PFD/Pluronic emul-sion caused an average decrease of 38%. As with interleukin-1β, production ofinterleukin-6 was abolished by lecithin and the lecithin/PFD emulsion. In thesesupernatants, the means are 157 pg/mL (lecithin) and 325 pg/mL (lecithin/PFDemulsion). In contrast, the supernatants of mononuclear leukocytes cultured onlywith culture medium and stimulated with LPS contained an average of44,239 pg/mL. None of the test compounds induced interleukin-6 production byitself.

Cell Proliferation Tests with ECV 304 and MonoMac6

The two human cell lines showed analogous behavior. The uptake of 3H-thymidine by the endothelial cells is not reduced in the presence of PFD andthe PFD/Pluronic emulsion (Tab. 2, Fig. 4). In the case of MonoMac6, the 3H-thymidine incorporation is slightly reduced. In contrast, the 3H-thymidine uptakeby endothelial and monocytic cells in the presence of lecithin and lecithin/PFDemulsion was nearly zero.

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ARTIFICIAL OXYGEN CARRIERS 65

Figure 3. Interleukin-6 release after incubation of mononuclear leucocytes with vari-ous test substances plus LPS. n = number of measurements, n = 4 for “none,” R.L., andPFD/lecithin; n = 2 for PFD and lecithin emulsion; n = 3 for Pluronic emulsion.

Cell Toxicity

The results of the cell toxicity assays are shown in Table 3. The measuredvalues for a incubation time of 2, 4, 6, and 8 h were within the controls. Ringer’slactate was again used as negative control, because the emulsions also contain

Table 2. Relative 3H-thymidine Incorporation of ECV 304 and MonoMac 6 AfterIncubation with Various Test Compounds (tc)

ECV 304 rel. 3H-thymidine MonoMac6 rel. 3H-thymidineTest Compound Incorporation [%]a Incorporation [%]a

Perfluorodecalin (PFD) 114 [73; 142] 84 [82; 87](n = 3)

Lecithin emulsion 1 [0; 2] 0 [0; 0](n = 3)

PFD/lecithin emulsion 2 [0; 4] 0 [0; 0](n = 3)

PFD/Pluronic emulsion 95 [81; 112] 87 [86; 88](n = 3)

100% 3H-thymidine incorporation: incubation of ECV 304 and MonoMac6 with culturemedium; n: number of measurements.aConfidence interval.

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66 DINKELMANN ET AL.

Figure 4. Relative 3H-thymidine incorporation of ECV 304 and MonoMac6 after incu-bation with various test compounds (tc); 100% 3H-thymidine incorporation = incubationof ECV 304 and MonoMac6 with culture medium.

it. The in vitro incubation of peripheral leukocytes itself leads to a slight in-crease in the rate of dead cells. After a 24-h incubation of leukocytes with PFDor PFD/Pluronic emulsion, about 20% dead monocytes and approximately 12%dead granulocytes were found. The lecithin/PFD emulsion was especially toxicfor monocytes (p < 0.005) and after 24 h the values for lymphocytes were also in-creasing. Figure 5 depicts the fraction of dead monocytes depending on incubationtime and test substance.

DISCUSSION

In our tests, perfluorodecalin alone did not cause hemolysis, but did causea slight suppression of interleukin-1β and interleukin-6 production and a lowtoxicity versus monocytes and granulocytes. PFD had only mild effects on cellproliferation of endothelial (ECV 304) and monocytic cells (MonoMac6).

Pluronic-based PFD emulsion caused mild to moderate inhibition ofendotoxin-induced cytokine production but showed no significant increase in toxic-ity compared to PFD. The influence of PFD /Pluronic emulsion on cell proliferationis low and comparable to PFD.

PFD-emulsion with lecithin as emulsifier suppressed the interleukin-1β/6release of LPS-stimulated monocytes to more than 98%. The lecithin-based PFDemulsion showed considerable toxicity for phagocytic cells, especially monocytes.Accordingly, the cell proliferation of endothelial and monocytic cells was alsonearly abolished.

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ARTIFICIAL OXYGEN CARRIERS 67

Tabl

e3.

Toxi

city

ofPe

rfluo

rdec

alin

(PFD

)an

dV

ario

usPF

DE

mul

sion

son

Peri

pher

alH

uman

Leu

cocy

tes

Frac

tion

ofD

ead

Cel

lsFr

actio

nof

Dea

dC

ells

Frac

tion

ofD

ead

Cel

lsIn

cuba

tion

Tim

e[h

]Te

stC

ompo

und

Am

ong

Lym

phoc

ytes

[%]

Am

ong

Mon

ocyt

es[%

]A

mon

gG

ranu

locy

tes

[%]

0R

inge

r’s

lact

ate

(n=

5)0

2±

10

PFD

(n=

7)0

4±

11

±1

PFD

/leci

thin

emul

sion

(n=

6)0

2±

11

±1

PFD

/Plu

roni

cem

ulsi

on(n

=4)

03

±2

013

Rin

ger’

sla

ctat

e(n

=5)

02

±1

1±

1PF

Dn.

t.n.

t.n.

t.PF

D/le

cith

inem

ulsi

on(n

=6)

2±

126

±7

2±

1PF

D/P

luro

nic

emul

sion

(n=

4)0

15±

128

±6

17R

inge

r’s

lact

ate

(n=

5)1

±1

5±

41

±1

PFD

(n=

7)1

±1

13±

74

±1

PFD

/leci

thin

emul

sion

(n=

6)3

±2

80±

21a

4±

4PF

D/P

luro

nic

emul

sion

(n=

4)1

±1

18±

614

±6

24R

inge

r’s

lact

ate

(n=

5)1

±1

6±

52

±1

PFD

(n=

7)1

±1

19±

1411

±5

PFD

/leci

thin

emul

sion

(n=

6)20

±11

82±

21a

14±

4PF

D/P

luro

nic

emul

sion

(n=

4)1

±1

21±

914

±6

ap

<0.

005,

n=

num

ber

ofm

easu

rem

ents

.

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68 DINKELMANN ET AL.

Figure 5. Toxicity of perfluorodecalin (PFD) and vaarious PFD emulsions on peripheralhuman monocytes.

In conclusion, PFD alone is generally untoxic. As PFD is not soluble inwater, it must be emulsified for intravenous application. We tested two emulsions,using either lecithin (2.5% w/v) or Pluronic (4% w/v) as emulsifier. The lecitihin/PFD emulsion showed considerable toxicity versus monocytes and inhibition ofimportant cellular functions (cytokine release and cell proliferation). This toxicitycannot be attributed to PFD but to the emulsifier or to the interactive effects of theentire PFD emulsion. In some of the tests we also used lecitihin as 2.5% (w/v)emulsion in Ringer’s lactate without other components. Lecithin alone suppressedthe cytokine release and the cell proliferation to the same extent as the PFD/lecithinemulsion. These results seem to show that lecithin was responsible for the immuno-suppressive effects. Edwards et al. (6) studied the effect of a similar PFD/lecithinemulsion on neutrophil chemoluminescence. The generation of a superoxide anionradical is another important neutrophil function. Edwards et al. (6) showed a dose-dependant decrease in PMA-induced neutrophil chemoluminescence. In contrastto our experiments, this effect could not be attributed to lecithin. Other authors(7,8) reported trombocytopenia in healthy adults after administration of low doses(∼1 mL/kg body weight) of PFC emulsions containing egg yolk lecithin.

The PFD/Pluronic PE 6800 emulsion tested showed, among other things,mild to moderate inhibition of endotoxin-induced cytokine production. The ef-fects and properties of Fluosol DA�, also a PFD emulsion containing Pluronic F68and perfluorotripropylamine, have been studied for years. Preclinical and clinicalstudies (9) have been undertaken to evaluate the properties and the benefits forpatients with acute blood loss (10), myocardial infarction (11), and those under-going percutanous transluminal coronary angioplasty (PTCA) (12), and so on. In

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ARTIFICIAL OXYGEN CARRIERS 69

animal experiments with dogs (13), it was shown that Fluosol is able to reducethe infarct size after reperfusion. The authors demonstrated in a following study(14) that the infusion of Fluosol resulted in a reduction in neutrophil concentrationafter 12–24 h, a suppression of neutrophil chemotaxis, and lysozyme release. Inthis way, Fluosol prevented, at least in part, the so-called myocardial reperfusioninjury. These immunsuppressive effects of Fluosol can also be detrimental. Micegiven Fluosol and endotoxin showed lower survival rates (15).

The immunosuppressive effects, especially of the lecithin-based PFD emul-sion we tested, might be beneficial if the emulsion is used in acute ischemia (e.g.,myocardial infarction) to diminish the postischemic inflammatory damage. Butit can have an unwanted immunosuppressive effect when used in high doses incombination with immunodeficiency or sepsis of the recipient.

In conclusion, our system includes a differentiated pattern of biocompatibil-ity assays, which in total provide an in vitro biocompatibility profile of the testedsubstances. Thus, it can be a useful tool in preclinical development of any oxygencarriers and other drugs for intravenous application.

REFERENCES

1. Remy, B.; Deby Dupont, G.; Lamy, M. Red Blood Cell Substitutes: Fluo-rocarbon Emulsions and Haemoglobin Solutions. Br. Med. Bull. 1999, 55,277–298.

2. Habler, O.; Kleen, M.; Messmer, K. Artificial Oxygen Carriers. Alternativesto Homologous Blood Transfusion? Zentralbl. Chir. 1999, 124, 260–270.

3. Spence, R.K.; Norcross, E.D.; Costabile, J.; McCoy, S.; Cernaianu, A.C.;Alexander, J.B.; Pello, M.J.; Atabek, U.; Camishion, R.C. Perfluorocarbonsas Blood Substitutes: The Early Years. Experience with Fluosol DA-20% inthe 1980s. Art. Cells Blood Subst. Immob. Biotechnol. 1994, 22, 955–963.

4. Takahashi, K.; Wasaki, Y.; Hata, J.; Mukai, K.; Goto, T. Spontaneous Trans-formation and Immortalization of Human Endothelial Cells. In Vitro 1990,25, 265–274.

5. Ziegler-Heitbrock, H.W.L.; Thiel, E.; Futterer, A.; Herzog, V.; Wirtz, A.;Riethmuller, G. Establishment of a Human Cell Line (Mono Mac 6) withCharacteristics of Mature Monocytes. Int. J. Cancer 1988, 41, 456–461.

6. Edwards, C.M.; Lowe, K.C.; Rohlke, W.; Geister, U.; Reuter, P.; Meinert,H. Effects of a Novel Perfluorocarbon Emulsion on Neutrophil Chemilumi-nescence in Human Whole Blood In Vitro. Art. Cells Blood Subst. Immob.Biotech. 1997, 25, 255–260.

7. Keipert, P.E. Use of Oxygent, a Perfluorochemical-Based Oxygen Carrier, asan Alternative to Intraoperative Blood Transfusion. Art. Cells Blood Subst.Immob. Biotechnol. 1995, 23, 381–394.

Art

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70 DINKELMANN ET AL.

8. Kaufman, R.J. The Results of a Phase I Clinical Trial of a 40% V/V Emulsionof HM (Oxyfluor). Art. Cells Blood Subst. Immobil. Biotechnol. 1994, 22,A112.

9. Ohyanagi, H.; Saitoh, Y. Development and Clinical Application of Perfluo-rochemical Artificial Blood. Int. J. Art. Organs 1986, 9, 363–368.

10. Gould, S.A.; Rosen, A.L.; Sehgal, L.R.; Sehgal, H.L.; Langdale, L.A.;Krause, L.M.; Rice, C.L.; Chamberlin, W.H.; Moss, G.S. Fluosol-DA asa Red-Cell Substitute in Acute Anemia. N. Engl. J. Med. 1986, 314, 1653–1656.

11. Wall, T.C.; Califf, R.M.; Blankenship, J.; Talley, J.D.; Tannerbaum, M.;Schwaiger, M.; Gacioch, G.; Cohen, M.D.; Sauz, M.; Leimberger, J.D. Intra-venous Fluosol in the Treatment of Acute Myocardial Infarction. Circulation1994, 90, 114–126.

12. Jaffe, C.C.; Wohlgelernter, D.; Cabin, H.; Bowman, L.; Dechelbaum, L.;Remetz, M.; Cleman, M. Preservation of Left Ventricular Ejection FractionDuring Percutaneous Transluminal Coronoary Angioplasty by Distal Tran-scatheter Coronary Perfusion of Oxygenated Fluosol DA 20%. Am. Heart J.1988, 6, 1156–1164.

13. Bajaj, A.K.; Cobb, M.A.; Virmani, R.; Gay, J.C.; Light, R.T.; Forman, M.B.Limitation of Myocardial Reperfusion Injury by Intravenous Perfluorochem-icals: Role of Neutrophil Activation. Circulation 1989, 79, 645–656.

14. Forman, M.B.; Pitarys, C.J., II; Vildibill, H.D.; Lambert, T.L.; Ingram, D.A.;Virmani, R.; Murray, J.J. Pharmacologic Perturbation of Neutrophils by Flu-osol Results in a Sustained Reduction in Infarct Size in the Canine Model ofReperfusion. J. Am. Coll. Cardiol. 1992, 19, 205–216.

15. Ohyanagi, H. Perfluorochemical Emulsions as Blood Substitutes: ClinicalData and New Applications. In Artificial Red Cells; Tsuchida, E., Ed.; JohnWiley and Sons: Chicester, 1995; 199–226.

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