It is said that the director of the Cancer Hospital has seen the "CAR-T solid tumor target hero spectrum: the brightest star in the night sky" since the last issue of Chen Bo, the chicken blood is full, and the hard-working map is strong to do CAR-T solid tumor research! Send a good job to Dr. Wang! Select the target! Design carrier! In vitro verification! In vivo experiment! Ethical! On the clinic! Be the dean! Welcome to the white and beautiful! Going to the peak of life!
However, half a year later, Xiong Director, Niu Niu, holding Chen Bo’s thighs and crying: “A CAR-T is like a sea. Since then, it’s a passerby.†“Is my experiment not smooth?†“For the CAR-T study Are pits everywhere?"
Chen Bo is also very helpless: "Old bear, you are too naughty, the lecture is not serious, in fact, many technical difficulties I have told you, today I will give you some inventory."
[First pit] Is your scFv sequence reliable?
Zi Yan: The target is easy to get, the antibody is difficult to screen! For common targets, the types of scFv sequences are vast, and the director Xiong is so overwhelmed.
How to choose between several tumor targets?
How to get the Shaf gold from the mixed fishes and scFv literature data and patent data?
How to get around the fake data of potholes?
How to find the kind of scFv with the highest affinity?
If you want to screen your own antibodies, what should you do?
Old bear, Jikai has cheats, hehe...
(For common target data, please refer to the article on April 21st of this public number: "CAR-T solid tumor target hero spectrum: the brightest star in the night sky" , which can be found in "View History News")
[Second pit] Is the quality of your CAR virus passing?
The director of the bear has the ability to carry the virus, but the quality and stability of the virus have encountered pits.
Not to mention that the basic tests such as mycoplasma, bacteria, fungi, and viral toxicity must be all negative, and the endotoxin <50 EU/ml must also meet the standard!
Old bear, do you think this is a qualified CAR virus? too young too simple! Is the T cell infected with the virus unstable? Can the animal experiment of CAR-T be repeated? Is your CAR virus purified by diafiltration? The total DNA residue should not exceed 10 ng/ul, and the BSA residue should not exceed 30 ng/ml! how about it? Are you afraid? !
In addition, even the Jikai version of the 屌 silk version of the lentivirus has been using QPCR absolute quantitative titer, the old bear is still using the inaccurate activity titer to dye T cells? Don't use the empty shell of the carrier to fool the T cells. !
Having said that, Chen Bo has been embarrassed to show off the photos of Jikai's GMP-class virus carrier production workshop. Let's think about the scenery.
(For detailed data, please refer to the article on April 8th of this public number: "How is the CarT Lentivirus on the tall, how it is made" , which can be found in "Viewing Historical News")
[Third pit] Do you think you will really separate PBMC?
Where do the T cells of preclinical experiments work? Director Xiong looked back at the bloodless Wang Bo: "Xiao Wang, come and come, and then smoke some blood~"
Old bear! Your doctor needs to save some use. I will first familiarize the experimental conditions of PBMC, otherwise I will not be able to recruit people next year!
Ficoll centrifugation should slow down and slow down, know?
Ficoll is toxic to T cells, and the total amount should be controlled.
Ficoll sucks too much and can cause PBMC to be difficult to wash.
Basic requirements for PMBC extraction: purity >90%, harvest rate >80%, live cell rate >95%, please treat every doctoral student! Cherish their blood and tears!
[Four pits] Classmates, your T cells stick to it?
Under the strict management of Director Xiong, Dr. Wang is in a sub-health state all the year round. The T cells that have been drawn out are also sluggish, and they have been raised for three days. They are all attached to the wall... The director of the study of solid tumors and the big eye of Dr. Wang Small eyes, suspended cells are so difficult to raise?
Lao Xiong, do you think that simply adding a little IL-2 to the culture medium is all right? Other cytokines are not satisfied!
Which serum effect is good to know?
Is the culture density of T cells clear?
Hey! The experimental operation can no longer be as extensive as a solid tumor!
[Fifth pit] Your T cell infection efficiency is å•¥ can not break through 50%?
The old bear sent Wang Bo to the hematology laboratory to study hard for a month of T cell culture Dafa, Wang Bo Xuecheng returned, and sure enough T cells were raised, good Kaisen! Good happi! CAR virus is dyed!
Huh? The infection efficiency is only 10%! Is it the wrong way to open it?
"Wang Bo, give it another dye!"
"Starting on the Holy Land, this time only 8%..."
"Xiao Wang, I am not giving you pressure, the infection efficiency can not be raised, you have to take more blood every month, the teacher is very worried about your health~"
"Mr. Bear... I am still a virgin, I don't want to die~~~"
"Look at the ink, go find Chen Bo and think of a way!"
On the other hand, Chen Bo gave Wang Bo a paper towel and comforted him: "Old Wang, you should come to me earlier. Jikai spent two years developing a set of CAR-T infection kits, top secret formula, T cells. The infection efficiency can reach 40-80%! Don't cry, just drink this bowl of brown sugar water"
[Sixth Pit] To be honest, how many times can your T cells expand?
However, Director Xiong is still too naive. It is not enough to improve the efficiency of T cell infection. The expansion factor in vitro is also the key!
How to effectively activate T cells?
How to amplify T cells by 25 times within 7 days?
What is the limit of in vitro expansion of T cells?
Did the grouping of CD3+ cells do it?
Did Jike's CAR-T activation amplification reagents have been tried?
How to control the morphological changes of T cells by activating reagents?
[Seventh pit] Is your control group reliable?
The control group is not a running dragon, and the CAR-T control group is more varied!
Targeting ability?
Killing ability?
Activation ability?
Proliferation ability?
Old bear, do you know which control group to choose?
What is the mutation principle of the CAR box sequence in the control group?
[The eighth pit] Is your animal model in the laboratory correct?
Bear director! Please hurry up your nude mouse! You can't continue to be embarrassed!
Please use NSG mice with more complete immunodeficiency! The mice were subjected to Prkdc and IL2rg double knockout mice directly on NOD mice using the CRISPR/Cas9 system. Theoretically, NSG mice did not have any background residue and the background was purer.
NSG mice lack mature T cells, B cells, and functional NK cells, lack cytokine signaling, and have almost no rejection of human T cells and tissues. There is also no B lymphocyte leakage. It is currently the internationally recognized tool for the most immunodeficiency and the most suitable for human cell transplantation.
In addition, NSG mice have an average life span of 1.5 years!
[The ninth pit] Are you really doing a CAR-T in vivo experiment?
Director Xiong, have you thought about it, how do animal models of solid tumors do?
Is the target cell added with a luciferase?
Subcutaneous tumor formation? Or is it a tail vein liver metastasis?
Does the circulatory system return to CAR-T?
Intratumoral or local injection?
When is the time to detect the killing effect of CAR-T?
How to detect the proliferation of CAR-T in vivo?
How long does it take for long-term testing?
[Twelfth pit] Can your time be spent?
The bear director who was hard-working and hard-working, overcame one technical pit, and finally eliminated the CAR-T preclinical experiment of tumor target X. The old bear who got the positive result was not happy at all. Three years! This project took a full three years!
In the past three years, Director Xiong’s head has become increasingly sparse, and Dr. Wang has repeatedly postponed... The most terrible thing is that the CAR-T article of the same target has been robbed, and the Principal of the next department took only one year to complete. Preclinical experiments with another target, ethics, clinical trials have been carried out...
The messy old bear in the wind silently walked onto the rooftop and lit a cigarette...
Due to limited space and words such as gold, Chen Bo can only pick a small part of the obvious pit today. The CAR-T basic experiment is undercurrent. Every pit has been experienced and painful. Nowadays, the paper is ridiculous, a bitter tear, I hope you can do it and cherish it.
After receiving the latest report from the production platform, Jikai’s “CAS9 Library Drug Genetic Screening†project has been officially released, and there will be a bloody hurricane on the rivers and lakes. Are you ready? And listen to the book "The high energy in the front: the nuclear weapons researched by the drug mechanism"
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