Methods for tissue embedding and tissue culturing

Methods of embedding and culturing tissue employing a fibrin glue composition of 2 to 100 mg/ml fibrinogen, 1 to 200 U/ml fibrinogen-activating enzyme, and 1 to 30 mM Ca(II) compound, are disclosed.

FIELD OF THE INVENTION 
This invention relates to methods of embedding and culturing tissue 
employing a fibrin glue composition comprising fibrinogen, 
fibrinogen-activating enzyme, and a calcium compound. 
BACKGROUND OF THE INVENTION 
The loss or failure of an organ or tissue can have a severe impact on the 
health and well-being of the affected person or animal. To understand the 
role of a particular organ or tissue in the body, how it grows, and its 
response to different agents, studies are done on samples of organ or 
tissue which are maintained in culture. The term "tissue" is used 
hereafter to refer to both organs and tissues. A problem in tissue culture 
studies is maintaining viability of the sample in culture. Another problem 
is the difficulty of routinely being able to prepare sections of tissue of 
a defined size for culturing. A lack of uniformity of the size of tissue 
sections means that accurate comparisons of results cannot be made. 
Tissue samples are usually embedded by fixing the tissue in formalin and 
then embedding in paraffin. Other fixatives that may be used to preserve 
tissue samples include mercuric chloride, picric acid, glutaraldehyde, 
Bouin's fluid, and Zenker's formalin (Helly's fluid). In addition to 
paraffin, plastic resins may be used for embedding the tissue samples. 
Disadvantages of these fixatives and embedding materials arise from trauma 
to the tissue, toxicity of the embedding material, and difficulty in 
obtaining sections of defined size, particularly of soft tissue. 
Fibrinogen is mitogenic and stimulates cell growth and viability. A human 
fibrin foam prepared by lyophilization of a solution of human fibrinogen 
has been used to culture tumors by placing pieces of tumor on top of 
10.times.10.times.3 millimeter semi-cubes of fibrin foam sitting in 
culture medium. Kalus, M. et al., Cancer 22:507-516 (1968); Kalus, M. et 
al., Arch. Path. 86:52-59 (1968). A disadvantage of this method is that 
only one side of the tissue sample is in contact with the fibrinogen, 
which minimizes the stimulatory effect of the fibrinogen. (All of the 
documents cited or otherwise referenced herein are incorporated herein in 
their entirety for all purposes.) 
Fibrin glue, usually made from freeze-dried plasma fibrinogen/factor XIII 
concentrate and thrombin, has been used in surgical applications such as 
replacement of sutures in skin grafts, nerve and vessel anastomoses, and 
surgery of parenchymal tissues of liver, lung, or spleen. In Itay U.S. 
Pat. Nos. 4,904,259 and 5,053,050, bone and cartilage are repaired by 
implanting a proliferating chondrocyte cell structure having phenotypic 
capability embedded in a biological glue consisting of 10-30% serum 
containing growth factors, 100 to 150 mg/ml fibrinogen, 60 to 90 U/ml 
thrombin, 60 mM CaCl.sub.2, and 2,000 U/ml (KIU) aproprotein. 
There is a continuing need for methods for embedding and culturing tissues 
that are non-toxic to the tissue, that allow improved sectioning of the 
tissue with less trauma to the tissue, and that allow better diffusion of 
tissue culture fluid to the sectioned tissue. 
SUMMARY OF THE INVENTION 
The methods of this invention satisfy those needs and have other advantages 
that will be apparent to those skilled in the art. The present invention 
provides methods of embedding and culturing tissue employing a fibrin glue 
composition comprising 2 to 100 milligrams/milliliter (mg/ml) fibrinogen, 
1 to 200 International Units/milliliter (U/ml) fibrinogen-activating 
enzyme, and 1 to 30 millimolar (mM) Ca(II) compound. As used herein, 
"Ca(II) compound" means a compound containing calcium in which the calcium 
nominally has a valence of +2, e.g., calcium chloride. The calcium 
compound can be any calcium salt. The fibrin glue composition can be used 
to embed tissue, which embedded tissue can be cut into sections 60 to 
1,000 microns thick, and to culture tissue. 
One advantage of the fibrin glue composition employed by the methods of 
this invention is that it is non-toxic to the tissue being embedded and/or 
cultured in it. Another advantage is that the fibrin glue composition 
imparts useful mechanical properties to the tissue embedded within it, 
which results in improved sectioning of the embedded tissue with less 
trauma to the embedded tissue. 
An advantage of the methods of embedding tissue in the fibrin glue 
composition is that the mechanical properties of the fibrin glue 
composition can be manipulated to provide the appropriate resistance to 
cutting for the type of tissue to be embedded and sectioned. 
The present invention also provides a method of culturing tissue embedded 
in the fibrin glue composition in which embedded tissue can accurately be 
cut into uniformly thick sections ranging in size from 60 to 1000 microns 
and the sections cultured. One advantage of this method is that the fibrin 
glue composition allows better diffusion of tissue culture fluid to the 
subsurface cells of the sectioned tissue, thereby minimizing tissue 
necrosis. Another advantage is that the fibrinogen and thrombin components 
of the fibrin glue, or their degradation products, are mitogenic and have 
a stimulatory effect on cell viability and growth of the cells of the 
sectioned tissue. 
Other features and advantages of this invention will be apparent to those 
skilled in the art.

DETAILED DESCRIPTION OF THE INVENTION 
In the present invention, tissue may be embedded using as a matrix a fibrin 
glue composition comprising 2 to 100 milligrams (mg) of fibrinogen per 
milliliter (ml) of glue composition, 1 to 200 International Units (U) of 
fibrinogen-activating enzyme per milliliter of glue composition, and one 
or more Ca(II) compounds in amounts sufficient to provide a calcium 
concentration in the glue composition of 1 to 30 millimolar (mM). Any 
tissue from any biological organism (e.g., human, non-human animal) may be 
used. The tissue can be fragments or complete organs such as heart or 
liver, tumors, or other types of tissue such as skin, or biopsy samples. 
Commercially available fibrinogen may be used in the fibrin glue 
composition and is available from Sigma Chemical Co., St. Louis, Mo. and 
Kabi, Stockholm, Sweden. Fibrinogen from fibrin sealant kits sold in 
Europe under the trade names Tisseel.TM. (Immuno, Austria) and 
Beriplast.TM. (Behringwerke, Marburg, Germany) may also be used. 
Cryoprecipitate fibrinogen prepared by blood banks may also be used in the 
fibrin glue composition. The fibrinogen is preferably human fibrinogen 
that has been virally inactivated (e.g., solvent/detergent-treated 
fibrinogen, hereafter "S/D fibrinogen"; see, e.g., U.S. Pat. Nos. 
4,540,573 and 4,764,369; Burnouf-Radosevich, M. et al., Vox Sang 58:79-84, 
"Biochemical and physical properties of solvent-detergent treated fibrin 
glue"). The mechanical properties of the fibrin glue and its resistance to 
cutting, i.e., breaking strength, may be adjusted to the tissue being 
embedded in the fibrin glue by altering the fibrinogen concentration. For 
soft tissues, such as liver, it is preferable to use fibrin glues with a 
low fibrinogen content of 2-20 mg/ml. For tough tissues, such as skin, it 
is preferable to use fibrin glues with a high fibrinogen content of 20-100 
mg/ml. By adjusting the mechanical properties of the fibrin glue to the 
tissue being embedded, better sections can be obtained with less trauma to 
the embedded tissue. 
The fibrinogen-activating enzyme may be thrombin, batroxobin, thrombocytin, 
ancrod, or other thrombin-like enzymes. Thrombin-like enzymes from snake 
venoms that coagulate fibrinogen are described in Seegers, W. H. & Ouyang, 
C., Handbook of Experimental Pharmacology 52:684-740 (1979), "Snake venoms 
and blood coagulation"; Stocker, K. et al., Thrombos. Diathes. Haemorrh. 
Suppl. 54:361-369 (1973)., "Reptilase as a defibrinogenating agent"; 
Niewiarowski, S. et al., Biochem. 18:3570-3577 (1979), "Thrombocytin, a 
serine protease from Bothrops atrox venom II"; Stocker K. et al., Toxicon. 
20:265-273 (1982), "Thrombin-like snake venom proteinases"; and Pirkle, H. 
et al., Thrombos. Haemostas. 65:444-450 (1991), "Thrombin-like enzymes 
from snake venoms: an inventory." It is preferred to use thrombin as the 
fibrinogen-activating enzyme, and more preferably human thrombin which has 
been virally inactivated (e.g., solvent-detergent-treated thrombin). 
When thrombin is the fibrinogen-activating enzyme, the preferred ranges for 
the components of the fibrin glue composition are 20 to 40 mg/ml 
fibrinogen, 2 to 20 U/ml thrombin, and 2 to 15 mM Ca(II) compound 
(preferably calcium chloride). 
Growth-modulating activity of the embedded tissue may also be modified or 
controlled by including biologically active materials in the fibrin glue 
matrix, such as growth factors, peptides, hormones, drugs, minerals, 
transition metal cations, or mixtures thereof. 
To embed the tissue, the tissue is preferably entirely immersed in the 
fibrin glue while the fibrin glue is in a fluid state. The length of time 
in which the fibrin glue will remain fluid is dependent upon the 
concentrations of fibrinogen and the fibrinogen-activating enzyme in the 
fibrin glue. When the fibrin glue sets, it forms a gel surrounding the 
tissue. The embedded tissue may then be sliced into sections that can 
range in size from 60 to 1000 microns. The sections can be maintained in 
suspension cultures or further encased in fibrin glue to surround all 
sides of the section with fibrin glue and then maintained in tissue 
culture medium or cryopreserved until ready to use as described in Fisher, 
R. et al., "Cryopreservation of pig and human liver slices," Cryobiology 
28:131-142 (1991). 
The fibrin glue may be prepared by premixing the fibrinogen with the Ca(II) 
compound, adding the tissue to be embedded, and then adding the 
fibrinogen-activating enzyme. After a suitable period of time, usually 
about one minute, this mixture may then be gently mixed and allowed to 
stand at room temperature for a suitable period of time, e.g., half an 
hour, until the resulting fibrin glue sets sufficiently and embeds the 
tissue. 
The fibrin glue may also be prepared by mixing fibrinogen and the Ca(II) 
compound with a low level of the enzyme (for example, thrombin), e.g., in 
the range of 0.5 to 2.0 U/ml, and then agitating the mixture mechanically 
or with air or nitrogen for about 15 seconds until there is much bubbling. 
The tissue to be embedded is then contacted with the glue mixture while 
the mixture is still fluid and bubbly, preferably by immersing the tissue 
into the glue mixture. Within a few minutes the bubbly mixture will set 
and form a fibrin glue foam around the tissue. The tissue encapsulated in 
the fibrin glue foam is then allowed to stand for a suitable period of 
time, e.g., one hour, at room temperature so that the glue foam completely 
sets and embeds the tissue. 
Regardless of how embedded in accordance with this invention, the embedded 
tissue can then be sliced using a tissue slicer, which is preferably 
maintained under sterile conditions. Embedding tissue in the fibrin glue 
of the present invention enables the tissue to be sliced to a thickness 
that permits the optimal diffusion of nutrients and gases within the 
tissue slice and thereby minimizes tissue necrosis. Section thickness of 
the fibrin glue embedded tissue may be varied from 60 to 1,000 microns. 
This thickness range allows good diffusion of nutrients and gases to the 
sub-surface cells during tissue culturing of the section. Diffusion of 
nutrients and gases is also aided by the open network in the fibrin glue. 
Section slices may be cultured under sterile conditions using standard 
tissue/organ culture methods, such as the method of Rheinwald, J. G., and 
Green. H. published in Cell, 6:331-334 (1975). Preferably the slices are 
transferred to multiwell petri dishes containing antibiotics and tissue 
culture medium appropriate for the tissue being cultured. Techniques which 
may be used for culturing organ slices are described in Smith, P. F. et 
al., Life Sciences 36:1367-75 (1985), "Dynamic organ culture of precision 
liver slices for in vitro toxicology"; and Connors, S. et al., "Evaluation 
of organic nephrotoxins using rabbit renal cortical slices", Alternative 
Methods in Toxicology, Vol. 6 (1988) (A .M. Goldberg, Ed.). Typically the 
cultures are incubated at 37.degree. C. under a CO.sub.2 incubator 
(95%-O.sub.2, 5%-O.sub.2) with gentle shaking. If the section slices are 
to be cultured at a later time, the section slices may be cryopreserved 
until such time. 
Alternatively, before placing a section slice made using this invention 
into tissue culture medium, the section slice may be further encased in 
fibrin glue using the embedding procedure described above so that all 
surfaces of the sliced tissue are covered with fibrin glue. The fibrin 
glue-encased tissue section may then be placed into suitable tissue 
culture medium and cultured as discussed above. 
To determine viability of the embedded tissue, a chromogenic substrate for 
plasmin is used. The tissue section is removed from the tissue culture 
medium and washed two times in saline, PBS, or tris buffer. If there has 
been cell growth and the section is viable, there will be a clear zone 
between the tissue and the surrounding fibrin glue due to fibrin lysis. 
Homminga, G. N. et al., Acta Orthop. Scand. 64:441-445 (1993), 
"Chondrocyte behavior in fibrin glue in vitro". This clear zone signifies 
secretion of plasminogen activator by viable cells. FIG. 1 shows a 
developed clear zone between the tissue and the surrounding fibrin glue. 
Alternatively, the washed section is incubated in S2251 substrate 
(H-D-Valyl-L-leucyl-L-lysine-p-nitroaniline dihydrochloride, available 
from Kabi, Stockholm, Sweden) and then checked for the appearance of a 
colored zone between the tissue and the surrounding fibrin glue. 
The following examples illustrate use of the present invention. 
EXAMPLE 1 
Embedding and Culturing of Tissue in Fibrin Glue 
The needle-affixing end of a sterile 10 ml plastic syringe (17 mm ID, 65 mm 
long) was cut off and the plunger was removed and set aside, leaving only 
the barrel. The cut end of the barrel of the syringe was sealed with 
stretched, moisture-proof, self-sealing, flat wrapper or film (e.g., 
AFILM.RTM. wrapper, American Can Packaging, Inc.) to prevent leakage 
when liquid was introduced. 
A 5.times.10 mm fragment of freshly excised mouse liver was washed in 
sterile saline. The liver fragment was placed into the syringe barrel onto 
the stretched film at the bottom of the syringe barrel. 2 ml Kabi Grade L 
fibrinogen (42 mg/ml) was poured into the syringe barrel so that the liver 
fragment was entirely immersed in the solubilized fibrinogen. 100 ul of 10 
U/ml thrombin with Ca(II) was added to the solution in the syringe barrel. 
The solution was gently mixed with a plastic Pasteur pipette two times and 
allowed to stand at room temperature for half an hour for the fibrin glue 
to gel and set. The purpose of stirring is to mix the components. 
The stretched film was removed and a 21 gauge syringe needle was inserted 
between the fibrin glue mass containing the liver fragment and the plastic 
syringe barrel to loosen any fibrin adhering to the barrel. The plunger 
was placed back into the top of the syringe barrel and then gently 
depressed to push out the cylindrical fibrin glue mass in which the liver 
fragment was embedded. 
Sections of the embedded liver fragment were cut from the fibrin glue mass 
utilizing a tissue slicer maintained under sterile conditions. The 
sections were 60-1000 microns thick. The sections were then transferred to 
a petri dish and maintained under standard tissue/organ culture conditions 
with Dubelco's modified Eagle Medium and 10,000 U penicillin and 
streptomycin in 5% CO.sub.2 in air at a temperature of 37.degree. C, with 
gentle shaking. 
Viability and cell growth were measured by monitoring the dissolution of 
the fibrin glue surrounding the sectioned liver fragment. Viability was 
measured daily using either a chromogenic substrate for plasmin or 
examining the section microscopically. Clear zones were found, 
respectively, around the cultured, sectioned, fibrin glue-embedded, skin 
fragment and heart fragment, indicating viability and cell growth of the 
sectioned organ fragments. 
EXAMPLE 2 
Alternative Method of Embedding and Culturing Of Tissue in Fibrin Glue 
The procedures of Example 1 were employed with the following variations. 
2 ml of fibrinogen (34 mg/ml) was poured into the syringe barrel and 
agitated mechanically (by vortex or shaking) for 15 seconds to induce 
bubbling. 
While this mixture was still fluid and bubbly, a 5.times.10 mm fragment of 
freshly excised and washed liver was dropped into the bubbly, still fluid 
mixture. Thereafter, thrombin and Ca(II) were added and mixed. After a few 
minutes, the bubbly mixture set and formed a fibrin glue foam. This fibrin 
glue foam was allowed to stand for one hour at room temperature. The 
fibrin glue foam may also be allowed to stand for one hour at 37.degree. 
C. 
After one hour, the fibrin glue foam containing the liver organ fragment 
was removed, sectioned, and cultured as described in Example 1. Viability 
was measured as previously described in Example 1. Microscopic examination 
indicated viability and growth. 
The above description is meant to be illustrative only of the present 
invention and not limiting thereof. Variations and modifications will be 
apparent to those skilled in the art and the claims are intended to cover 
all modifications and variations falling within the true spirit and scope 
of the invention.