BACKGROUND Bovine serum has been widely used in cell culture, and is classified into fetal bovine serum, newborn calf serum and fetal calf serum. Bovine serum provides not only hormones involved in cell growth and function but also adhesive factors and diffusive factors as well as proteins carrying hormones, heavy metals, lipids, etc. Fetal bovine serum has been most widely used of all bovine serum

since it stimulates the growth of almost all the cell lines and primary culture cells. However, in order to obtain fetal bovine, which is essential for the preparation of fetal bovine serum, comparatively a long period of fertilization (280 days) is required. Moreover, just 1.5 Q of fetal bovine serum is produced in a fetal bovine, suggesting low productivity and poor economical efficiency.

Newborn calf serum is inferior to fetal bovine serum in its effect on cell growth, but happens to be selected because it is superior to fetal calf serum and has better economical efficiency.

While bovine serum for cell culture is being produced, mycoplasma and some of viruses can be removed owing to the improved serum sterilizing techniques. Nevertheless, the safety of bovine serum has been in question ever since bovine spongiform encephalopathy and v. Creutzfeldt-Jakob disease (vCJD) of human was reported to be related to bovine serum. Thus, bovine serum only produced in specific countries where bovine spongiform encephalopathy has not been reported yet is now of use for the production of a vaccine or a biological medicine. But, if bovine spongiform encephalopathy is developed in even such countries, the supply of

bovine serum necessary for the production of a vaccine or a biomedicine must be dropped, resulting in a rising of prices. So, it is an urgent need to develop a substance that can be replaced with bovine serum.

Considering economical efficiency and safety, efforts have been made to develop a serum-free medium in order to culture cells without a serum or an additive which is, at least, helpful for reducing the amount of a serum consumed. However, the additives stimulating cell growth are expensive and the price goes much higher when the additives are added enough to induce satisfactory cell proliferation, which is as expensive as a serum.

Since the preparation method of a chicken embryo extract using a 10 day-old chicken embryo was first reported in 1966, all the researches with a chicken embryo extract have been accomplished by using a 10 to 12 day-old chicken embryo so far. The conventional method for the preparation of a chicken embryo extract is composed of emulsifying a 10 to 12 day-old chicken embryo, adding the same amount of phosphate buffered saline thereto in order to elute effective ingredients and obtaining supernatant

separated by centrifugation. The conventional chicken embryo extract stimulates the proliferation of nerve cells, muscle cells, blood stem cells and epithelial cells of pancreas. But, it is also known that an ingredient inhibiting the proliferation of fibroblasts is included in macromolecular fraction of the chicken embryo extract (Coogan et al. , 1968, Lab Invest, 18: 139-142). Birds are far away from mammals evolutionarily, so that the protein structure of a bird is far different from that of a human. Besides, unlike a cow, a bird has not had any disease that is fatal to a human, suggesting that a chicken embryo extract is comparatively safe.

The average egg-laying ratio of a chicken is 90%, comparing to the impregnation ratio of a cow.

So, during 280 impregnation days of a cow, a chicken lays 252 eggs (280 x 0.9). In addition, massed raising is possible for chickens, resulting in better productivity than a cow. A 10-day-old chicken embryo weighs 2-3 g, but it gains weight so fast from then on that it weighs 26-28 g right before hatching (20-21 days). Therefore, the conventional method using 10-12 day-old chicken embryos seems to have problems of low productivity, unsuitableness for the culture of fibroblasts, and

poor cell reproduction, comparing to a fetal bovine serum.

So, the present inventors prepared a chicken embryo extract using 18 day-old-prehatching stage embryos instead of using 10-12 day-old embryos. The present inventors further completed this invention by confirming that the chicken embryo extract of the invention greatly enhanced cell proliferation to the similar or superior level to the case using a fetal bovine serum and promoted productivity during the cell culture, comparing to the conventional chicken embryo extract.

SUMMARY OF THE INVENTION It is an object of this invention to provide a chicken embryo extract for cell culture obtained from 18 day-old to prehatching stage embryos.

It is also an object of this invention to provide a method for preparation of the above chicken embryo extract.

It is a further object of this invention to provide an additive for cell culture medium prepared by adding a serum and an effective ingredient to the above chicken embryo extract.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS In order to achieve the above object of the invention, the present invention provides a chicken embryo extract for cell culture obtained from 18 day-old to prehatching stage embryos.

The present invention also provides a method for preparation of the above chicken embryo extract.

The present invention further provides an additive for cell culture medium prepared by adding a serum and an effective ingredient to the above chicken embryo extract.

Further features of the present invention will appear hereinafter.

The present invention provides a chicken embryo extract for cell culture obtained from 18 day-old to prehatching stage embryos.

The conventional chicken embryo extract is prepared from 10 to 12 day-old chicken embryos. On the other hand, the chicken embryo extract of the present invention is characterized by being prepared from 18 day-old to prehatching stage embryos.

Comparing to a fetal bovine serum, the conventional chicken embryo extract has poor cell proliferative activity and low productivity because of its a light weight. However, the chicken embryo extract of the present invention shows excellent cell proliferative activity and much improved productivity owing to its, increased weight since the extract was prepared from 18 day-old to prehatching stage embryos.

In the preferred embodiment of the present invention, the productivity of each extract taken from embryos was measured, as they got old. 10 day- old chicken embryos weighed 2 g and 21 day-old chicken embryos weighed 25.7 g, showing almost 13 times weight increase, suggesting that the productivity of an extract taken from 21 day-old chicken embryos could be 13 times as high as that of an extract taken from 10 day-old chicken embryos (see Table 1). The fibroblast proliferative activity of chicken embryo extracts taken as the embryos got old (taken on day 10,14, 16,18, 21) was compared with those of a new born calf serum and a fetal bovine serum. As a result, when chicken embryo extracts taken from 10 day-old and 14 day-old

embryos were added during the cell culture, the proliferation and the adhesion to an incubator of fibroblasts were not observed, yet some of nerve cells and muscle cells were adhered to the incubator and proliferated a little. The adhesion and the proliferation were much less when a newborn calf serum was used than when chicken embryo extracts taken from 10 day-old and 14 day-old embryos were used. However, when chicken embryo extracts taken from over 16 day-old embryos were added during the cell culture, comparatively a large amount of fibroblasts were adhered to the incubator to form a layer, and the proliferation was as high as when a fetal bovine serum was used (see FIG. 1).

The proliferative activity of each conventional chicken embryo extract (taken from 10 to 12 day-old embryos), the chicken embryo extract of the present invention (taken from 18 day-old to prehatching stage embryos) and a fetal bovine serum was compared by MTT assay. As a result, regarding the activity of a fetal bovine serum as 100%, the conventional chicken embryo extract showed a 72% relative activity and the chicken embryo extract of the present invention showed a 86% relative activity, suggesting that the extract of the present invention

clearly had enhanced proliferative activity.

Further, when the chicken embryo extract of the present invention was added double, a 95% relative activity was measured, suggesting that the extract was much superior to a fetal bovine serum in the economical aspect (see Table 2).

The proliferative activity of the conventional chicken embryo extract (taken from 10 to 12 day-old embryos), the chicken embryo extract of the present invention (taken from 18 day-old to prehatching stage embryos) and a fetal bovine serum to chicken embryo liver cells was also compared. As a result, regarding the activity of a fetal bovine serum as 100%, the conventional chicken embryo extract showed a 105% relative activity and the chicken embryo extract of the present invention did a 126% relative activity, suggesting that the extract of the present invention had much increased proliferative activity (p<0.05) to liver cells, comparing to the conventional chicken embryo extract (see Table 3).

In addition, the adhesion and the proliferative activity of the chicken embryo extracts taken from 10,14, 16,18, and 21 day-old embryos were observed using a kidney cell line, which were then compared with those of a new born calf serum and a fetal

bovine serum. As a result, the extracts taken from 10,14, and 16 day-old embryos showed poor adhesion to an incubator and low proliferative activity. In the meantime, when the chicken embryo extracts taken from 18 day-old and 21 day-old embryos were added to the cell line, the cell adhesion to the incubator was clearly observed to form a layer and the proliferation was enhanced as well (see FIG. 2).

And, the effect of the chicken embryo extract of the present invention taken from 18 day-old to prehatching stage embryos on the kidney cell proliferation was investigated. As a result, the cell proliferative activity of the extract of the present invention was superior to that of a conventional chicken embryo serum. Further, the proliferative activity of the chicken embryo serum of the present invention was as excellent as or superior to that of a fetal bovine serum (see Table 4-Table 7).

Therefore, the chicken embryo extract of the present invention, extracted from 18 day-old to prehatching stage embryos, is superior to a conventional chicken embryo extract (taken from 10 to 12 day-old embryos) in productivity and in cell

proliferative activity.

The present invention also provides a preparation method of the above chicken embryo extract consisting of the following steps: 1) Emulsifying 18 day-old to prehatching stage embryos; and 2) Eluting effective ingredients by adding a buffer solution to the emulsified chicken embryo.

In the above step 1), it is preferable to use a solvent selected from a group consisting of phosphate buffered saline, saline and balanced salt solution to emulsify embryos, but the choice is not always limited thereto. In the preferred embodiment of the present invention, phosphate buffered saline was used as a solvent.

In the above step 2), it is preferable to add a buffer solution selected from a group consisting of phosphate buffered saline, saline and balanced salt solution. In the preferred embodiment of the present invention, phosphate buffered saline was used as a buffer solution. It is also preferable to add a buffer solution by the same amount with the volume of the emulsified chicken embryo.

The present invention further provides an additive for cell culture medium prepared by adding a serum or an effective ingredient to the above chicken embryo extract.

The additive for cell culture medium of the present invention was prepared by adding a serum or an effective ingredient to the chicken embryo extract obtained above, by which cell proliferation was much enhanced, comparing to when the chicken embryo extract of the invention was used alone.

The said serum was preferably selected from a group consisting of a bovine serum, a horse serum, a swine serum, a rabbit serum and a chicken serum, but not always limited thereto.

The said effective ingredient can be one of a vitamin, a mineral, an amino acid, a hormone, a growth factor or a mixture thereof, but the choice is not always limited thereto.

BRIEF DESCRIPTION OF THE DRAWINGS The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

FIG. 1 is a photograph of cell culture showing the proliferative activities of chicken embryo extracts taken from chicken embryos on schedule to chicken embryo fibroblasts.

FIG. 2 is a photograph of cell culture showing the proliferation of a PK-15 swine kidney cell line that was cultured after being added with each chicken embryo extract taken from chicken embryos on schdule or a fetal bovine serum by 10%.

EXAMPLES Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Preparation of chicken embryo extracts <1-1> Comparison of each weight gained as an embryo is growing and of the productivity of each extract

10 to 21 day-old chicken embryos were aseptically emulsified. The same amount of phosphate buffered saline (or Hank's BSS) was added thereto. Effective ingredients were eluted at room temperature or in cold storage for over 1 hour, and supernatant was obtained by centrifugation. The obtained supernatant was filtered, resulting in the preparation of a chicken embryo extract (Coon, H. G. and Cahn, R. D., Science, 153: 1116-1119,1966 ; Paul, J."Cell and Tissue Culture. "Edinburgh, Churchill Livingstone, pp. 172-184,1975).

As a result, the chicken embryo weighed 2 g on day 10, and 25.7 g on day 21, showing almost 13 times increased weight. Thus, the productivity of an extract prepared from 21 day-old chicken embryos was 13 times as high as that of another extract prepared from 10 day-old chicken embryos (Table 1).

<Table 1> Day 10 12 14 16 18 21 Weight (g) 2 5 7. 6 13. 6 20. 8 25.7 Extract 2 5 7. 6 13. 6 20. 8 25.7 (m)

Example 2: The proliferative activity of the chicken embryo extract to fibroblasts <2-1> Comparison of the adhesion and the proliferative activities of conventional chicken embryo extract and the chicken embryo extract of the present invention to chicken embryo fibroblasts The proliferative activities of conventional chicken embryo extract and the novel chicken embryo extract of the present invention to chicken embryo fibroblasts were investigated. Precisely, the 11 day-old chicken embryos were cut elaborately, and 0.25% trypsin was added, followed by digestion at 37 C twice for 10 minutes. The digested cells were filtered by twofold gauze, followed by centrifugation. After the centrifugation, chicken embryo fibroblasts were washed with phosphate buffered saline (pH 7.2) twice. The cell number was adjusted to 1.5 x 103/well, which was then cultured in a 6-well culture plate. The culture medium used was prepared by mixing 199 medium (GIBCO-BRL, NY, USA) and F10 medium (GIBCO-BRL, NY, USA) at the rate of 1: 1. Each chicken embryo extract, a newborn calf serum (NCS, GIBCO-BRL, NY, USA) or a fetal bovine serum (FBS, GIBCO-BRL, NY, USA) was added to the

culture medium by 10% concentration. During the culture, the adhesion and the proliferation of chicken embryo fibroblasts were observed with a microscope.

As a result of an observation at 16th hour and the 36th hour from the beginning of the culture, the fibroblasts, cultured on the medium in which chicken embryo extract taken from 10 day-old or 14 day-old chicken embryos was added, were not adhered to the bottom of the plate, and just some of muscle cells and nerve cells were adhered to the bottom of the plate and proliferated. On the other hand, the fibroblasts, cultured on the medium in which chicken embryo extract taken from 16 day-old, 18 day-old or 21 day-old chicken embryos was added, were adhered evenly to the bottom of the plate and proliferated thereon. In the meantime, the fibroblasts cultured on the medium in which a new born calf serum was added, was least adhered or proliferated. The fibroblasts cultured on the medium in which a fetal bovine serum was added, showed similar activity to that cultured on the medium in which chicken embryo extract taken from over 16 day-old chicken embryos was added (FIG. 1).

Therefore, a novel chicken embryo extract of the present invention was confirmed to have best cell adhesion and proliferative activity to chicken embryo fibroblasts.

<2-2> Comparison of the growth of chicken embryo fibroblasts stimulated by conventional chicken embryo extract and the chicken embryo extract of the present invention The growth of chicken embryo fibroblasts each stimulated by conventional chicken embryo extract (taken from 10-12 day-old chicken embryos), by the chicken embryo extract of the present invention (taken from 20 day-old chicken embryos) and by a fetal bovine serum was compared. The chicken embryo fibroblasts, prepared in the above Example <2-1>, were cultured in a 96-well plate by 2 x 102/well.

Each chicken embryo extract, a newborn calf serum (GIBCO-BRL) or a fetal bovine serum (GIBCO-BRL) was added to the culture medium by 10 % concentration.

Three days later, medium was removed, and the numbers of live cells were quantified by MTT assay.

Particularly, each well was supplemented with 50 go of MTT solution (MTT 0.5% aqueous solution) and 20 M of culture medium, and then cultured at 37 C for

1-3 hours. 100 M of DMSO was added thereto, followed by shaking for 30 minutes. Finally, OD562 was measured with an ELISA reader. The mean values of OD562 of each well, respectively supplemented with conventional chicken embryo extract, the chicken embryo extract of the present invention and a fetal bovine serum, were calculated. Statistical significance was investigated by paired T-test.

Regarding the OD of a fetal bovine serum as 100%, relative activities (%) of each extract and serum were measured (Mathematical Formula 1).

Mathematical Formula 1> Relative activity (%) = mean OD562 of chicken embryo extract/mean OD562 of FBS x 100 As a result, a relative activity of the chicken embryo extract of the present invention was 86%, and a relative activity of the conventional extract was 72%, suggesting that the novel extract of the present invention had higher growth stimulating activity (p<0.05) than the conventional extract.

Moreover, when the chicken embryo extract of the

present invention was added double (20%), a relative activity reached 95%, suggesting that the novel extract of the present invention was more beneficial and economical than a fetal bovine serum (Table 2).

<Table 2> Conventio EmbEx EmbEx FBS nal (10%) (20%) (10%) chicken embryo extract (100) 0.143 0.186 0.206 0.184 0. 154 0. 169 0. 212 0. 223 0. 157 0. 174 0. 217 0. 223 0. 153 0. 206 0. 250 0. 203 0. 167 0. 186 0. 219 0. 224 0. 204 0. 174 0. 172 0. 193 0. 181 0. 176 0. 186 0. 219 0. 149 0. 174 0. 212 0. 216 0.130 0.176 0.168 0.214 OD562 0.186 0.181 0.215 0.228 0. 157 0. 175 0. 230 0. 232 0. 157 0. 187 0. 205 0. 190 0. 192 0. 175 0. 199 0. 199 0. 154 0. 180 0. 217 0. 221 0. 122 0. 194 0. 196 0. 239 0. 141 0. 202 0. 212 0. 241 0. 139 0. 185 0. 186 0. 223 0. 140 0. 241 0. 208 0. 233 Mean OD 0. 157 0. 186 0. 206 0. 217 Relative 72% 86% 95% 100% activity

In the above <Table 2>, EmbEx means the chicken embryo extract of the present invention and FBS means a fetal bovine serum.

Example 3: Ability of each chicken embryo extract to stimulate and to maintain the growth of chicken embryo liver cells The ability of each chicken embryo extract to stimulate and to maintain the growth of chicken embryo liver cells was investigated. First, 14 day- old chicken embryo liver tissues were collected aseptically, which was digested at 4°C for 6 hours with 0. 05% trypsin. The digested cells were filtered by the same method as used for the culture of chicken embryo fibroblasts, and then washed by centrifugation in order to separate liver cells.

The media were prepared by being supplemented with a newborn calf serum (NBCS), a fetal bovine serum (FBS), and chicken embryo extracts taken from 10 day-old and 21 day-old chicken embryos by 10% respectively. The chicken embryo liver cells were

inoculated in a 96-well culture plate (5x102 cells/well) and cultured. In order to investigate the proliferative capacity of the liver cells, the numbers of live cells were counted by MTT on the 3rd day of culture. The mean values of ODgga of each well, respectively supplemented with conventional chicken embryo extract, the chicken embryo extract of the present invention and a fetal bovine serum, were calculated. Statistical significance was investigated by paired T-test. Regarding the OD of a fetal bovine serum as 100%, relative activities (%) of each extract and serum were measured.

As a result, a relative activity of the conventional chicken embryo extract was 105%, and a relative activity of the chicken embryo extract of the present invention was 126% (p<0.05) (Table 3).

Therefore, the chicken embryo extract of the present invention was confirmed to have much enhanced activity, comparing to a fetal bovine serum.

<Table 3> Conventional EmbEx FBS chicken (10%) (10%) embryo extract (10%) 0.325 0.406 0.343 0. 363 0. 590 0. 360 0. 359 0. 420 0. 372 0. 402 0. 456 0. 332 0. 359 0. 458 0. 344 0. 371 0. 434 0. 346 0. 395 0. 402 0. 348 0.376 0.445 0.354 OD562 0.348 0.436 0.352 0. 341 0. 447 0. 339 0.372 0.406 0.343 0.372 0.438 0.341 0. 358 0. 413 0. 332 0. 353 0. 395 0. 340 0. 325 0. 399 0. 330 0. 354 0. 414 0. 322 Mean OD 0. 360 0. 435 0. 344 Relative 105% 126% 100% activity

Example 4: The effect of chicken embryo extract on the proliferation of kidney cell lines The growth stimulating activity of the chicken embryo extract of the present invention to kidney cell lines was investigated. As a kidney cell line, a swine kidney originated cell line (PK-15), a bovine kidney originated Mardin-Darby bovine kidney

cell line (MDBK), a canine kidney originated Mardin- Darby canine kidney cell line (MDCK), a green monkey kidney originated cell line (Vero) were used. In order to prepare culture medium, a conventional chicken embryo extract, the chicken embryo extract of the present invention and a fetal bovine serum were added into RPMI 1640 respectively by 10%. The above each cell line was inoculated into a 96-well culture plate (3x102 cells/well) and cultured.

Observation under a microscope was done at 4th hour and on 3rd day of culture, and the adhesion and the proliferation were investigated by MTT.

As a result, in the case of culturing PK-15 cell line with a fetal bovine serum or the chicken embryo extract of the present invention, it was observed at 4th hour that the cell morphology was changed from a round shape to a pyramid shape, which was adhered to the bottom of the culture plate, and on the 3rd day, a layer almost evenly formed on the bottom of the culture plate was seen. The cells cultured with the chicken embryo extract of the present invention grew faster and better than the cells cultured with conventional chicken embryo extract and than those cultured with a fetal bovine

serum, too (FIG. 2). The cell proliferation was also investigated by MTT assay. As a result, the conventional chicken embryo extract showed a 89% relative activity, comparing to a fetal bovine serum, and the novel chicken embryo extract of the present invention showed a 114% relative activity (p<0.05) (Table 4). Therefore, the chicken embryo extract of the present invention was confirmed to stimulate the proliferation of kidney cell lines better than a fetal bovine serum.

<Table 4> Conventional EmbEx FBS chicken (10%) (10%) embryo extract (10%) OD562 0.225 0. 301 0. 288 0. 263 0. 478 0. 293 0. 259 0. 332 0. 311 0. 302 0. 361 0. 286 0. 259 0. 347 0. 279 0. 271 0. 343 0. 299 0. 295 0. 311 0. 285 0. 276 0. 337 0. 295 0. 248 0. 328 0. 321 0. 241 0. 339 0. 283 0. 272 0. 312 0. 301 0. 272 0. 328 0. 280 0.258 0.321 0.297 0. 253 0. 305 0. 287 0. 225 0. 298 0. 299 0. 254 0. 313 0. 303 Mean OD 0. 261 0. 335 0. 294 Relative 89% 114% 100% activity

The capacity of the extract of the present invention to stimulate the proliferation of MDBK, MDCK, Vero cell lines was also investigated. As a result, the chicken embryo extract of the present invention showed a 157% relative activity in MDBK cell line, and a conventional chicken embryo extract did a 90% relative activity therein (Table 5).

Likewise, 94% and 80% in MDCK cell line (Table 6), and 101% and 83% each in Vero cell line (Table 7).

Thus, the chicken embryo extract of the present invention was proved to have enhanced proliferative activity, comparing to a conventional chicken embryo extract. In addition, the chicken embryo extract of the present invention was confirmed to be superior to a fetal bovine serum in its activity and in the economical aspect.

<Table 5> Conventional EmbEx FBS chicken (10%) 10% ##) embryo extract (10%) 0.144 0.327 0.184 0. 144 0. 345 0. 228 0. 145 0. 276 0. 139 0. 184 0. 348 0. 207 0. 229 0. 342 0. 232 0. 216 0. 317 0. 180 0. 217 0. 378 0. 256 0. 209 0. 378 0. 251 0. 236 0. 365 0. 167 OD562 0. 194 0. 351 0. 309 0. 215 0. 360 0. 241 0. 198 0. 351 0. 184 0. 186 0. 362 0. 247 0. 238 0. 372 0. 258 0. 255 0. 387 0. 252 0. 142 0. 373 0. 287 0. 243 0. 396 0. 253 0. 253 0. 355 0. 192 Mean OD 0. 203 0. 355 0. 226 Relative 90% 157% 100% activity <Table 6> Conventional EmbEx FBS chicken (10%) (10%) embryo extract (10%) 0.557 0.571 0.604 0. 481 0. 576 0. 709 0. 470 0. 612 0. 643 0. 601 0. 566 0. 636 0. 511 0. 663 0. 740 0. 630 0. 626 0. 635 0. 502 0. 561 0. 738 0. 563 0. 694 0. 722 0.475 0.677 0.629 OD562 0.562 0.627 0.637 0. 492 0. 708 0. 645 0. 569 0. 659 0. 591 0. 643 0. 680 0. 655 0. 737 0. 719 0. 506 0. 562 0. 661 0. 501 0. 867 0. 660 0. 656 Mean OD 0. 534 0. 627 0. 668 Relative 80% 94% 100% activity <Table 7> Conventional EmbEx FBS chicken (10%) (10%) embryo extract (100) Oases 0.462 0.652 0.874 0. 574 0. 689 0. 579 0. 482 0. 691 0. 731 0.627 0.718 0.934 0. 584 0. 786 0. 700 0. 662 0. 771 0. 667 0. 540 0. 653 0. 657 0. 527 0. 667 0. 615 0. 562 0. 687 0. 695 0. 520 0. 673 0. 563 0. 544 0. 656 0. 722 0. 622 0. 647 0. 587 0. 542 0. 690 0. 694 0. 631 0. 682 0. 553 0. 448 0. 545 0. 624 0. 565 0. 528 0. 478 Mean OD 0. 556 0. 671 0. 667 Relative 83% 101% 100% activity

INDUSTRIAL APPLICABILITY As explained hereinbefore, the chicken embryo extract of the present invention taken from 18-day old to prehatching stage embryos can be effectively used for cell culture because it has a similar cell proliferative activity to a bovine serum and high productivity as well, but no dangerous factor causing bovine spongiform encephalopathy.

Those skilled in the art will appreciate that

the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.